1. Kumvetsetsa koyamba
Pakadali pano, tikuyenera kumvetsetsa malingaliro ndi mawu ofotokozera, kuti tipewe kulakwitsa pamaso pa akuluakulu athu, monga:
Q: Kodi pali kusiyana kotani pakati pa RT-PCR, qPCR, Real-time PCR, ndi RT-PCR yeniyeni?
Yankho: RT-PCR ndi reverse transcript PCR(reverse transcription PCR, RT-PCR), yomwe imagwiritsidwa ntchito kwambiri ndi polymerase chain reaction (PCR).Mu RT-PCR, chingwe cha RNA chimasinthidwa kukhala DNA yowonjezera, yomwe imagwiritsidwa ntchito ngati template yokulitsa DNA ndi PCR.
Real-time-PCR ndi qPCR(Quantitative Real-ltime-PCR) ndi chinthu chomwecho, onsewa ndi nthawi yeniyeni yochuluka ya PCR, zomwe zikutanthauza kuti kuzungulira kulikonse kwa PCR kumakhala ndi zolemba zenizeni zenizeni, kotero chiwerengero cha ma templates oyambira chikhoza kusinthidwa kusanthula kolondola.
Ngakhale onse a Real-time PCR (real-time fluorescent quantitative PCR) ndi Reverse transcript PCR (reverse transcription PCR) akuwoneka kuti amafupikitsidwa ngati RT-PCR, msonkhano wapadziko lonse ndi: RT-PCR ikutanthauza kumasulira mobwereraPCR , Real-time PCR nthawi zambiri amafupikitsidwa ngati qPCR (kuchuluka kwa nthawi yeniyeni PCR).
Ndipo RT-PCR (RT-qPCR) yanthawi yeniyeni, Ndi PCR yolembera kumbuyo yophatikizidwa ndi ukadaulo wochulukirachulukira.: choyamba pezani cDNA (RT) kuchokera ku RNA reverse transcript, ndiyeno gwiritsani ntchito Real-time PCR pakuwunika kochulukira (qPCR).Ma laboratories ambiri amachita RT-qPCR, ndiye kuti, kafukufuku wa RNA mawu pansi-regulation, kotero kuti qPCR yomwe aliyense amakamba mu labotale imanena za RT-qPCR, koma musaiwale kuti pali mayeso ambiri a DNA pamagwiritsidwe azachipatala.Kusanthula kachulukidwe, monga kuzindikira kachilombo ka hepatitis B HBV.
Funso: Pambuyo powerenga zambiri za PCR ya fulorosenti, chifukwa chiyani chidutswa chokulitsa chiyenera kuyendetsedwa mkati mwa 80-300bp?
Yankhani: Kutalika kwa mndandanda uliwonse wa jini ndi wosiyana, ena ndi ma kb angapo, ena ndi mazana a bp, koma timangofunika kuti kutalika kwa mankhwala kukhale 80-300bp popanga zoyambira, zazifupi kapena zazitali kwambiri sizoyenera kudziwa za fulorosenti ya PCR.Chidutswa cha mankhwalawa ndi chachifupi kwambiri kuti chisiyanitsidwe ndi primer-dimer.Kutalika kwa primer-dimer ndi pafupifupi 30-40bp, ndipo n'zovuta kusiyanitsa ngati ndi primer-dimer kapena mankhwala ngati ndi osachepera 80bp.Ngati chidutswa cha chinthucho ndi chachitali kwambiri, choposa 300bp, chidzapangitsa kuti pakhale kutsika kwapamwamba kwambiri ndipo sichikhoza kuzindikira bwino kuchuluka kwa jini.
Mwachitsanzo, powerenga kuti m’kalasi muli anthu angati, mumangofunika kuwerengera pakamwa pa anthu angati.N'chimodzimodzinso mukamazindikira majini, mumangofunika kuzindikira mndandanda wina wa jini kuti muyimire Mndandanda wonsewo udzachita.Ngati mukufuna kuwerenga anthu, muyenera kuwerenga pakamwa ndi mphuno, makutu, ndi magalasi, ndipo n’zosavuta kulakwitsa.
Kukulitsa, mu kafukufuku wa zamoyo, pali zochitika zambiri zofufuza kuchokera kumalo kupita kumalo, chifukwa mndandanda wamtundu wamtundu uliwonse ndi wautali kwambiri, sikofunikira komanso kosatheka kuyeza zidutswa zonse, monga kutsatizana kwa bakiteriya 16S, zomwe ndikuchita ndondomeko yokhazikika ya mabakiteriya Assays kuti afotokoze chiwerengero cha mabakiteriya ena.
Q: Kodi mulingo woyenera kutalika kwa qPCR pulayimale mapangidwe?
Yankhani: Nthawi zambiri, kutalika koyambira ndi pafupifupi 20-24bp, zomwe zili bwino.Zachidziwikire, tiyenera kulabadira mtengo wa TM wa choyambira popanga choyambira, chifukwa izi zimagwirizana ndi kutentha koyenera kwa annealing.Pambuyo poyesera zambiri, zatsimikiziridwa kuti 60 ° C ndi mtengo wabwino wa TM.Ngati kutentha kwa annealing kuli kotsika kwambiri, kungayambitse kukulitsa kosagwirizana kwenikweni.Ngati kutentha kwa annealing ndikokwera kwambiri, mphamvu yokulitsa idzakhala yocheperako, nsonga ya mapindikidwe okulitsa idzayamba pambuyo pake, ndipo mtengo wa CT udzachedwa.
Q: Kodi njira ya utoto imasiyana bwanji ndi njira ya kafukufuku?
Yankho: Njira ya utotoUtoto wina wa fulorosenti, monga SYBR Green Ⅰ, PicoGreen, BEBO, ndi zina zotero, sutulutsa kuwala paokha, koma umatulutsa fluorescence pambuyo pomangika ku kabowo kakang'ono ka DNA yazingwe ziwiri.Choncho, kumayambiriro kwa machitidwe a PCR, makinawo sangathe kuzindikira chizindikiro cha fulorosenti.Zomwe zimachitika zikafika pagawo lokulitsa, chingwe chachiwiri chimatsegulidwa, ndipo chingwe chatsopano chimapangidwa pansi pa DNA polymerase, ndipo molekyulu ya fulorosenti imamangiriza ku dsDNA kakang'ono poyambira.Pamene kuchuluka kwa ma PCR akuwonjezeka, utoto wochulukirapo umaphatikizidwa ndi DNA yamitundu iwiri, ndipo chizindikiro cha fulorosenti chimakulitsidwanso mosalekeza.Njira yopangira utoto imagwiritsidwa ntchito kwambiri pofufuza zasayansi.
PS: Samalani poyesa, utoto uyenera kuphatikizidwa ndi DNA yamunthu, samalani kuti musinthe kukhala munthu wa fulorosenti.
Njira yopaka utoto (kumanzere) Njira yofufuzira (kumanja)
PS: Samalani poyesa, utoto uyenera kuphatikizidwa ndi DNA yamunthu, samalani kuti musinthe kukhala munthu wa fulorosenti.
SYBR Green Ⅰ imamangiriza ku kanjira kakang'ono ka DNA
Njira yofufuzaTaqman probe ndiye njira yomwe imagwiritsidwa ntchito kwambiri ndi hydrolysis.Pali gulu la fulorosenti kumapeto kwa kafukufuku wa 5′, nthawi zambiri FAM, ndipo kafukufukuyo pawokha amatsatana ndi jini yomwe mukufuna.Pali gulu lozimitsa fulorosenti kumapeto kwa 3′.Malinga ndi mfundo ya fluorescence resonance mphamvu kutengerapo (Förster resonance mphamvu kutengerapo, FRET), pamene mtolankhani fulorosenti gulu (wopereka fulorosenti molekyulu) ndi quenching fulorosenti gulu (kulandira fulorosenti molekyulu) amasangalala Pamene sipekitiramu zikuphatikizana ndi mtunda uli pafupi kwambiri (7-10 excitation kuvomereza molekyulu canceres the molekyulu) , pamene autofluorescence imafooka.Choncho, kumayambiriro kwa machitidwe a PCR, pamene kafukufukuyo ali omasuka komanso osasunthika mu dongosolo, gulu la mtolankhani fulorosenti silidzatulutsa fulorosisi.Pamene annealing, choyambira ndi probe kumangiriza kwa template.Panthawi yowonjezera, polymerase imapanga maunyolo atsopano mosalekeza.DNA polymerase ili ndi 5'-3 ′ exonuclease ntchito.Ikafika pa kafukufukuyo, DNA polymerase idzatulutsa kafukufuku kuchokera ku template, kulekanitsa gulu la mtolankhani fulorosenti ku gulu la quencher fulorosenti, ndikumasula chizindikiro cha fulorosenti.Popeza pali mgwirizano umodzi ndi umodzi pakati pa kafukufuku ndi template, njira yofufuzira ndi yopambana kuposa njira ya utoto pokhudzana ndi kulondola ndi kukhudzidwa kwa mayesero.Njira yofufuzira imagwiritsidwa ntchito kwambiri pozindikira.
Q: Kodi quantification mtheradi ndi chiyani?Kodi Relative Quantification ndi chiyani?
Yankhani: Kuchulukitsidwa kotheratu kumatanthawuza kuwerengera nambala yoyambirira yachitsanzo chomwe chiyenera kuyesedwa ndi qPCR, monga ma virus angati a HBV omwe ali mu 1ml ya magazi.Chotsatira chomwe chimapezedwa ndi kuchuluka kwachibale ndikusintha kwa kuchuluka kwa jini yomwe mukufuna mumtundu wina wokhudzana ndi chitsanzo china, ndipo mawonekedwe a jini amawongolera kapena kutsika.
Q: Kodi kuchuluka kwa RNA m'zigawo, kubweza bwino zolembera, komanso kukulitsa bwino kungakhudze zotsatira zoyeserera?
Q: Kodi kusungirako zitsanzo, ma reagents otulutsa, zosinthira zolembera, ndi zotumizira zotumiza zopepuka zimakhudza zotsatira zoyeserera?
Q: Ndi njira iti yomwe ingakonzere deta yoyesera?
Pankhani izi, tidzazifotokoza mwatsatanetsatane m'magawo apamwamba komanso apamwamba pansipa.
2. Chidziwitso chapamwamba
Pankhani ya nthawi yeniyeni ya fulorosenti yochuluka ya PCR, tiyenera kuzindikira kuti masauzande a mapepala ofufuza asayansi amasindikizidwa chaka chilichonse, pakati pawo luso la fulorosenti la PCR silochepa.
Ngati palibe muyezo wamba woyezera kuyezetsa kwa fulorosenti ya PCR, zotsatira zake zitha kusiyanasiyana.Kwa jini yomweyi yamtundu womwewo, wokhala ndi njira yopangira yofanana, zotsatira zodziwikiratu zidzasiyananso mosiyanasiyana, ndipo zidzakhala zovuta kwa ochedwa kubwereza zotsatira zomwezo.Inu Palibe amene akudziwa chomwe chili chabwino ndi cholakwika.
Kodi izi zikutanthauza kuti fluorescent quantitative PCR ndiukadaulo wachinyengo kapena ukadaulo wosadalirika?Ayi, ndichifukwa choti fulorosenti yochulukirachulukira ya PCR imakhala yozindikira komanso yolondola kwambiri, ndipo kachitidwe kolakwika pang'ono kamatulutsa zotsatira zosiyana.Kutayika pang'ono ndi makilomita chikwi.Wolemba nkhaniyo akhoza kuzunzidwa mobwerezabwereza ndi obwereza.Panthawi imodzimodziyo, owunikiranso magazini amakhalanso ovuta kusankha kuchokera ku zotsatira zosiyana zoyesera.
Zonsezi, zikuwonetsa kusowa kwa mgwirizano pakuyesa zenizeni za PCR.Kuti izi zitheke, asayansi akulu mumakampani adayamba kupanga miyezo,ikufuna opereka chithandizo kuti apereke zina zofunika zoyeserera ndi kukonza deta (kuphatikiza zofunikira) m'nkhaniyo kuti akwaniritse izi.
Owunika akhoza kuweruza ubwino wa kuyesera powerenga izi;owerenga amtsogolo angagwiritsenso ntchito izi kubwereza kuyesa kapena kukonza zoyeserera.Ndiye zotsatira zoyesera zomwe zapezedwa motere ndizodzaza ndi chidziwitso, zapamwamba, komanso zogwiritsidwa ntchito.
MIBBI (Zochepa Zochepa Zofufuza Zachilengedwe ndi Zachilengedwe -http://www.mibi.org) idakhalapo.MIBBI ndi pulojekiti yomwe imapereka miyezo yoyesera.Zimafalitsidwa m'chilengedwe.Pulojekitiyi imayang'ana zoyeserera zosiyanasiyana zachilengedwe, kuphatikiza cell biology, Microarray, qPCR yomwe tikambirana pano, ndi zina zambiri, ndipo imapereka mtundu uliwonse wa kuyesa popereka zolembedwa pamanja.Chidziwitso chimenecho chiyenera kuperekedwa nthawi zonse.
Mu projekiti ya MIBBI, pali zolemba ziwiri zokhudzana ndi fulorosenti kuchuluka kwa PCR, zomwe ndi:
·RDML (Real-Time PCR Data Markup Language) - chinenero cholongosoka ndi kalozera wa malipoti a nthawi yeniyeni ya deta ya PCR;
·MIQE (Chidziwitso Chochepa Chofalitsa Mayesero a PCR a Quantitative Real-Time) - mfundo zochepa zofalitsa nkhani zokhudzana ndi kuyesa kwa PCR mu nthawi yeniyeni.
Choyamba, tiyeni tikambirane za RDML, mawu akuti terminology.
Ngati palibe kutanthauzira koyenera kwa chirichonse, sikutheka kupitiriza kukambirana, chifukwa chake kufotokozera mawu ndikofunika kwambiri pa mayeso.
Mawu ogwiritsidwa ntchito poyesa fulorosenti ya PCR ali ndi izi.QIAGEN watipangira chidule chabwino kwambiri.Zotsatirazi zonse ndi zoumakatundu .
Amplification curve
Mapiritsi a amplification amatanthawuza kupindika komwe kumapangidwa panthawi ya PCR, ndi nambala yozungulira ngati abscissa ndi mphamvu yanthawi yeniyeni ya fluorescence panthawi yakuchita ngati ordinate.
Mapindikira abwino kwambiri amplification ayenera kukhala ndi izi: maziko ake ndi athyathyathya kapena akuchepa pang'ono, ndipo palibe chowonekera chokwera;malo opindika a curve ndi omveka bwino, ndipo kutsetsereka kwa gawo la exponential kumayenderana ndi kukulitsa bwino.Kuchulukira kotsetsereka, kumapangitsanso kukulitsa bwino;curve yonse yokulitsa Kufanana ndikwabwino, kuwonetsa kuti kukulitsa kwachubu chilichonse kuli kofanana;gawo la exponential la amplification curve of low-concentration samples likuwonekera.
Zoyambira (zoyambira)
Choyambira ndi mulingo waphokoso wa kuzungulira koyambirira, yomwe nthawi zambiri imayezedwa pakati pa 3rd ndi 15th cycle, chifukwa kuwonjezeka kwa mtengo wa fluorescence chifukwa cha kukulitsa sikungathe kudziwika panthawiyi.Chiwerengero cha mikombero chomwe chimagwiritsidwa ntchito powerengera choyambira chikhoza kukhala chosiyanasiyana ndipo chingafunike kuchepetsedwa ngati kuchuluka kwa ma template kumagwiritsidwa ntchito kapena ngati mulingo wa jini yowunikirayo uli wapamwamba.
Kukhazikitsa maziko kumafuna kuyang'ana deta ya fluorescence kuchokera ku mzere wokulitsa curve.Zoyambira zimayikidwa kuti kukula kwa curve yokulitsa kumayamba ndi nambala yozungulira yokulirapo kuposa nambala yoyambira yoyambira.Mizere yoyambira iyenera kukhazikitsidwa payekhapayekha pazotsatira zomwe mukufuna.Makhalidwe amtundu wa fluorescence omwe amapezeka m'mizere yoyambirira ayenera kuchotsedwa pamtengo wa fluorescence womwe umapezeka muzinthu zokulitsa.Mitundu yaposachedwa ya mapulogalamu osiyanasiyana a Real-Time PCR amalola kukhathamiritsa kwapang'onopang'ono kwa zisankho zapayekha.
M'mizere ingapo yoyambirira ya kukulitsa kwa PCR, chizindikiro cha fluorescence sichisintha kwambiri.Kuyandikira mzere wowongoka kumatchedwa maziko, koma ngati tiyang'ana mozama pazigawo zingapo zoyambirira, tikuwona kuti mkati mwazoyambira ndi zomwe zikuchitika pachithunzichi.
Background Background amatanthauza
mtengo wosagwirizana ndi fluorescence pamachitidwe .Mwachitsanzo: kuzimitsa fluorescence kosakwanira;kapena chiwerengero chachikulu cha ma templates a DNA omwe ali ndi mizere iwiri chifukwa chogwiritsa ntchito SYBR Green.Zigawo zakumbuyo za chizindikirocho zimachotsedwa mwamasamu ndi algorithm ya pulogalamu ya Real-Time PCR.
Mtolankhani chizindikiro
Chizindikiro cha mtolankhani chimanena za siginecha ya fulorosenti yopangidwa ndi SYBR Green kapena ma probe omwe amalembedwa motsatizana nthawi ya Real-Time PCR.
Normalized Reporter Signal (RN)
RN imatanthawuza mphamvu ya fulorosenti ya utoto wa mtolankhani wogawidwa ndi mphamvu ya fulorosenti ya utoto wodziyimira pawokha womwe umayesedwa nthawi iliyonse.
Passive Reference Dye
M'ma PCR a Nthawi Yeniyeni,utoto wa fulorosenti ROX umagwiritsidwa ntchito ngati kalozera wamkati kuti asinthe chizindikiro cha fulorosenti.Imawongolera kusiyanasiyana chifukwa cha kuyika kwa mapaipi olakwika, malo abwino, ndi kusinthasintha kwa fluorescence pamaziko abwino.
Chiyambi cha fluorescence (chiyambi)
idasinthidwa pamwamba pa mtengo wakumbuyo komanso pansi kwambiri pamtengo wamapiri a amplification curve.Iyenera kukhala mumzere wozungulira wa curve yokulirapo, kuyimira mndandanda wamtundu wa PCR.Ma threshold akhazikike mu mawonekedwe a log-amplification curve kuti gawo la log-linear la PCR lidziwike mosavuta.Ngati pali ma jini angapo mu Real-Time PCR, malire akuyenera kukhazikitsidwa pa chandamale chilichonse .Nthawi zambiri, chizindikiro cha fulorosenti choyamba 15 m'zinthu PCR anachita ntchito ngati fulorosenti maziko chizindikiro, ndi fluorescence pakhomo ndi 10 nthawi muyezo kupatuka kwa fulorosenti chizindikiro choyamba 3 kuti 15 m'zinthu PCR, ndi fluorescence pachimake pa gawo la amponplential wa PCR.Mwambiri, chida chilichonse chimakhala ndi malire ake a fluorescence omwe amayikidwa musanagwiritse ntchito.
Cycle Threshold (CT) kapena Crossing Point (CP)
Kuzungulira komwe kokhotakhota kumadutsa pakhomo (ie, pomwe kuzindikira kwa fluorescence kumawonjezeka kwambiri).CT ikhoza kukhala kachigawo kakang'ono ndipo kuchuluka kwa template yoyambira kumatha kuwerengedwa.Mtengo wa CT umayimira kuchuluka kwa mikombero yomwe siginecha ya fulorosenti mu chubu chilichonse cha PCR imafika poyambira.Pali mgwirizano wa mzere pakati pa mtengo wa CT wa template iliyonse ndi logarithm ya nambala yoyambira ya template,kukwera nambala yoyambira, yocheperako mtengo wa CT, ndi mosemphanitsa.Mzere wokhotakhota ukhoza kupangidwa pogwiritsa ntchito mulingo wokhala ndi nambala yodziwika yoyambira, momwe abscissa imayimira mtengo wa CT, ndipo wolumikizira amayimira logarithm ya nambala yoyambira.Choncho, malinga ngati mtengo wa CT wa chitsanzo chosadziwika ukupezeka, chiwerengero choyambirira cha chitsanzocho chikhoza kuwerengedwa kuchokera pamzere wokhazikika.
Mtengo wa ΔCT
ΔCT mtengo umafotokozakusiyana pakati pa jini yomwe mukufuna ndi jini yofananira yamtundu wa CT, monga jini yosamalira m'nyumba, ndipo imagwiritsidwa ntchito kukonzanso kuchuluka kwa template yomwe imagwiritsidwa ntchito:
⇒ΔCT = CT (jini yomwe mukufuna) - CT (jini yodziwika bwino)
Mtengo wa ΔCT
Mtengo wa ΔΔCT umafotokoza kusiyana pakati pa tanthauzo la ΔΔCT lachitsanzo cha chidwi (mwachitsanzo, maselo olimbikitsidwa) ndi tanthauzo la ΔΔCT lachitsanzo (mwachitsanzo, maselo osasunthika).Zitsanzo zomwe zimatchulidwanso zimatchedwanso chitsanzo cha calibration ndipo zitsanzo zina zonse zimasinthidwa kuti zikhale zofanana:
⇒ΔCT = pafupifupi ΔCT (chitsanzo cha chidwi) - pafupifupi ΔCT (chitsanzo chofotokozera)
Ma genes osatha (endogenous reference genes)
Mawonekedwe amitundu yodziwika bwino, monga majini osamalira m'nyumba (majini osamalira m'nyumba), samasiyana pakati pa zitsanzo.Kuyerekeza mikhalidwe ya CT ya jini yolozera ku jini yomwe mukufuna kumalola kuti mulingo wa jini womwe mukufuna kuti ukhale wokhazikika ku kuchuluka kwa RNA kapena cDNA (onani gawo la ΔCT pamwamba).
Ma genes amkati olondolazotheka kuwonongeka kwa RNA kapena kukhalapo kwa ma enzyme inhibitors mu zitsanzo za RNA, komanso kusiyanasiyana kwa zomwe zili mu RNA, reverse transcript performance, nucleic acid recovery, and sample handle.Kuti tisankhe jini yolondolera bwino lomwe, tidasintha ma aligorivimu kuti alole kusankha kwake koyenera kutengera zoyeserera.
Ulamuliro wamkati
Njira yowongolera yomwe imakulitsidwa m'njira yofanana ndi ndondomeko ya chandamale ndikufufuzidwa ndi kafukufuku wina (ie, kuchita duplex PCR).Zowongolera zamkati nthawi zambiri zimagwiritsidwa ntchito poletsa zokulitsa zolephereka, monga ngati kutsata kwa chandamale sikudziwika.
Calibration Chitsanzo
Chitsanzo (mwachitsanzo, RNA yoyeretsedwa kuchokera ku mzere wa selo kapena minofu) yogwiritsidwa ntchito poyerekezera ndi zitsanzo zina zonse kuti zizindikire msinkhu wa jini.Chitsanzo choyezera chikhoza kukhala chitsanzo chilichonse, koma nthawi zambiri chimakhala chowongolera (mwachitsanzo, chitsanzo chosasamalidwa kapena chitsanzo kuchokera pa nthawi ya zero).
Zowongolera zabwino
gwiritsani ntchito zowongolera ndizodziwika kuchuluka kwa template.Kuwongolera kwabwino nthawi zambiri kumagwiritsidwa ntchito poyang'ana ngati seti yoyambira kapena seti ya probe ikugwira ntchito bwino komanso kuti zomwe zimachitikazo zakhazikitsidwa bwino.
No Template Control (NTC)
Dongosolo loyang'anira lomwe lili ndi zigawo zonse zofunika za kamvekedwe kakukulitsa kupatula template, yomwe nthawi zambiri imasinthidwa ndi madzi.Kugwiritsa ntchito NTC kumatha kupeza kuipitsidwa komwe kumachitika chifukwa cha kuipitsidwa kwa reagent kapena DNA yakunja, motero kuwonetsetsa kutsimikizika ndi kudalirika kwa data yozindikira.Kukula kwa ulamuliro wa NTC kumasonyeza kuipitsidwa.
Palibe RT control (NRT)
Njira yochotsera RNA ikhoza kukhala ndi DNA yotsalira ya genomic, yomwe ili yovulaza kwambiri ndipo ndiyomwe imakhudza mtundu wa data komanso mdani wachilengedwe wa qPCR, kotero popanga zoyeserera, ziyenera kupangidwa kuti zizingokulitsa kuzindikira kwa RNA.Pali njira ziwiri, imodzi ndiyo kupanga zoyambira kudutsa ma introns, ina ndikuchotsatu DNA, yomwe ili yabwinoko, yomwe tidzakambirana pambuyo pake.Kuwongolera kwa NTR ndi galasi lamatsenga kuti lizindikire kuipitsidwa kwa DNA.Ngati pali kukulitsa, zikutanthauza kuti pali kuipitsa.
Miyezo
Miyezo ndi zitsanzo za ndende yodziwika bwino kapena nambala yojambulidwa yomwe imagwiritsidwa ntchito popanga ma curve wokhazikika.Pofuna kuonetsetsa kukhazikika kwa muyezo, gawo la jini nthawi zambiri limapangidwa mu plasmid ndikugwiritsidwa ntchito ngati muyezo.
Mzere wopindika
nthawi zambiri amachepetsedwa kukhala osachepera 5 ndende zowerengera ndi mankhwala muyezo malinga ndi kuwirikiza chiŵerengero, ndi 5 mfundo kukokedwa mu makonzedwe a CT mtengo ndi kukopera nambala, ndi mfundo zolumikizidwa kupanga mzere kupanga pamapindikira muyezo.Pamapindikira aliwonse okhazikika, kutsimikizika kwake kuyenera kuyang'aniridwa.Mtengo wotsetsereka umagwera pakati pa -3.3 ndi -3.8, ndipo ndende iliyonse imachitidwa katatu.Mfundo zomwe ndi zosiyana kwambiri ndi mfundo zina ziyenera kutayidwa.Mtengo wa CT wa chitsanzo kuti uyesedwe umabweretsedwa muzitsulo zokhazikika, ndipo mlingo wa mawu a chitsanzo kuti uyesedwe ukhoza kuwerengedwa.
Mtengo wa CT wa chitsanzo kuti uyesedwe umabweretsedwa muzitsulo zokhazikika, ndipo chiwerengero choyambirira cha chitsanzo choyesedwa chikhoza kuwerengedwa.
Kuchita bwino ndi Kutsetsereka
Kutsetsereka kwa curve yokhazikika kumayimira mphamvu ya PCR yeniyeni.
·Kutsetsereka kwa -3.322 kumasonyeza kuti kukulitsa kwa PCR ndi 1, kapena 100% kothandiza, ndipo kuchuluka kwa mankhwala a PCR kumawirikiza kawiri panthawi iliyonse.
Kutsetsereka kochepera -3.322 (mwachitsanzo, -3.8) kumasonyeza mphamvu ya PCR
·Kutsetsereka kwakukulu kuposa -3.322 (mwachitsanzo, -3.0) kumasonyeza kuti mphamvu ya PCR ikuwoneka yokulirapo kuposa 100%, zomwe zimachititsa chidwi, kodi mkombero umodzi wa PCR ungapangitse bwanji kuchulukitsa kuwirikiza kawiri?Izi zimachitika mu gawo losagwirizana la PCR, ndiko kuti, pali kuchuluka kwakukulu kosagwirizana kwenikweni.
kusungunuka kopindika
Mukamaliza kukulitsa kwa qPCR, chinthu cha PCR chimatenthedwa.Kutentha kumakwera, chinthu chokulitsa mizere iwiri chimasungunuka pang'onopang'ono, zomwe zimapangitsa kuchepa kwa mphamvu ya fluorescence.Kutentha kwina (Tm) kukafika, zinthu zambiri zimasungunuka.Fluorescence imatsika kwambiri.Zogulitsa zosiyanasiyana za PCR zimakhala ndi ma Tm osiyanasiyana komanso kutentha kosiyana kosungunuka, kuti mawonekedwe a PCR adziwike.
Mpiringidzo wosungunuka (mzere wotuluka)
Njira yosungunuka imapangidwa kuti ipange mapu apamwamba, omwe amatha kuwonetsa bwino momwe zidutswa zazinthu za PCR zilili.Popeza kutentha kosungunuka ndi mtengo wa Tm wa chidutswa cha DNA, magawo ena omwe amakhudza mtengo wa Tm wa chidutswa cha DNA akhoza kuweruzidwa, monga kukula kwa fragment, GC content, ndi zina zotero.kutalika kwa mankhwala okulitsa kumakhala pakati pa 80-300bp, kotero kutentha kosungunuka kuyenera kukhala pakati pa 80 ° C ndi 90 ° C.
Kutanthauzira kopindika kosungunuka: Ngati nsonga yaikulu yokha ikuwoneka pakati pa 80 ° C-90 ° C, zikutanthauza kuti fulorosenti yochuluka ya PCR ndi yangwiro;Ngati nsonga yaikulu ikuwoneka pakati pa 80 ° C-90 ° C ndi nsonga zosiyanasiyana zikuwonekera pansi pa 80 ° C, primer dimer imaganiziridwa.Mukhoza kuyesa kuonjezera kutentha kwa annealing kuti muthetse;ngati nsonga yaikulu ikuwonekera pakati pa 80 ° C-90 ° C, ndipo nsonga yosiyana siyana ikuwonekeranso pamene kutentha kumakwera, makamaka kumaganiziridwa kuti pali kuipitsidwa kwa DNA, ndipo DNA iyenera kuchotsedwa pa gawo loyambirira la kuyesa.
Zoonadi, pali zochitika zina zachilendo, zomwe zidzagawidwe m'munsimu.
3. Chidziwitso chapamwamba
Kuti ndichite qPCR, ndiyenera kunena MIQE,Zambiri Zochepakwa Publication ofZambiriNthawi Yeniyeni PCRKuyesera -chidziwitso chocheperako pakusindikiza zolemba zenizeni - nthawi yochuluka ya PCRzoyesera .Kuti tifewetse kumvetsetsa kwa aliyense, tifewetsa mfundo zazikuluzikulu.
Mutha kusaka zolemba zoyambirira za MIQE pa intaneti, ndipo chofunikira kwambiri ndi chakutimndandanda wa data womwe umayenera kuperekedwa pofalitsa nkhani .
Owunika akhoza kuweruza ubwino wa kuyesera powerenga izi;owerenga amtsogolo angagwiritsenso ntchito izi kubwereza kapena kukonza zoyeserera.
Ndizofunikira kudziwa kuti pamndandandawu, kufunikira kwa mndandanda uliwonse kumalembedwa ndi E kapena D motsatana.Zikutanthauza chiyani?E: zambiri zofunika (ziyenera kutumizidwa);D: chidziwitso chofunikira (perekani momwe mungathere).
MIQE (1)—Mapangidwe Oyesera
Ambiri a scumbags omwe amaliza chitetezo chawo akamaliza maphunziro awo omaliza maphunziro sangadziwe kupanga kuyesera payekha, kutsegula zolemba zawo, ndikuchita zomwe mphunzitsi amawauza kuti achite.Chotsatira chake, mapangidwe oyeserawo sanali okhwima, ndipo dipatimenti yokonza magaziniyi inanena kuti akufuna kupanga chithunzi ichi ndi chithunzicho, kotero adachichita modzidzimutsa.Umu ndi momwe zinyalala zimapangidwira!
Pafupi ndi kunyumba, mfundo yoyamba yoyesera ndiyo kudziwakukhwima kwa logic yoyesera.Chinthu chofunika kwambiri ndi mapangidwe oyesera, ndipo chofunika kwambiri pakupanga kuyesera ndi momwe mungakhazikitsire chitsanzo chandamale, chitsanzo chowonetsera (kuwongolera), ndi chiwerengero cha kubwerezabwereza, kotero kuti deta yoyesera ikhoza kutchulidwa, kufananizidwa, ndi kukhutiritsa.
Chitsanzo chandamaleamatanthauza chitsanzo chomwe chimafuna kuti tizindikire jini yomwe tikufuna pambuyo pa chithandizo china.Chitsanzo chofotokozerandiye chitsanzo popanda chithandizo chilichonse, chomwe nthawi zambiri chimatchedwa mtundu wakutchire mu biology.
Zoyeserera zoyesererandi zofunika kwambiri.Kawirikawiri, chiwerengero cha obwerezabwereza okopa ayenera kukhala oposa atatu.Ndikofunikira kusiyanitsa kuti kubwereza kwachilengedwe ndi chiyani komanso kubwereza kwaukadaulo.
Biological Replicates: Kuyesera komweko kotsimikizira kochitidwa ndi zinthu zosiyanasiyana (nthawi, zomera, magulu, mbale zochitira).
Kubwerezabwereza kwachilengedwe
Tiyeni titenge chitsanzo cha mankhwala a tsabola.Tikufuna kupopera mankhwala pa zomera zitatu za ABC, ndiye kuti zomera zitatu za ABC ndizofanana ndi zamoyo zitatu, ndipo ndizoyesa kutsimikizira komweko kochitidwa ndi zipangizo zosiyanasiyana.Koma monga kuyesa, kuwongolera kumafunikadi, kotero kuti tikhoza kupopera imodzi mwa nthambi za chomera A kupanga gulu loyesera la chomera A, osati kupopera mbewu zina za chomera A kupanga gulu lolamulira.Chitani zomwezo kwa B ndi C.
Zofananira Zaukadaulo (Zojambula Zaukadaulo): Ndi kuyesa kobwerezabwereza kopangidwa kuti tipewe zolakwika zomwe zimachitika chifukwa cha ntchito, zomwe kwenikweni ndi bowo lobwereza lomwe limaphatikizidwa muzinthu zomwezo.Njira zonse zochiritsira ndi zowongolera ziyenera kukhala ndi makonda obwereza (osachepera atatu) amtundu womwe mukufuna komanso jini yolozera mkati.
Kubwereza kwaukadaulo
Tengani tsabola wothiridwa ndi mankhwala monga chitsanzo kachiwiri.Pagulu loyesera la chomera A, tidapanga mabowo atatu a PCR a 1, 2, ndi 3 pa jini yake yomwe akufuna komanso jini yolozera mkati motsatana, kuti titenge avareji pambuyo podziwika.Kuwongolera mbewu A Magulu amathandizidwanso chimodzimodzi.Mofananamo, chitani mankhwala omwewo kwa zomera B ndi C.Uku ndikubwereza kwaukadaulo.
Ndikoyenera kuzindikira zimenezozomwe zimalowa m'mawerengerowo ndi kubwerezabwereza kwachilengedwe, ndipo kubwereza kwaukadaulo ndikoyesa ngati pali zochitika zongochitika mwachisawawa poyeserera, kuti zotsatira zoyeserera zikhale zodalirika, ndiko kuti, kupewa zolakwika potenga pafupifupi pafupifupi momwe timanenera nthawi zambiri.
Zowongolera Zoyipa—NTC ndi NRT
NTC (No-Template Control), chiwongolero chopanda template, chimagwiritsidwa ntchito kutsimikizira ngati zinthu zoyeserazo zili ndi kachilombo.Kawirikawiri, madzi amagwiritsidwa ntchito ngati template.Ngati pali fluorescent reaction, zimasonyeza kuti nucleic acid kuipitsidwa kwachitika mu labotale.
Kuipitsa kumeneku kumachokera ku: madzi odetsedwa, ma reagents osayenerera okhala ndi DNA yokhazikika, kuyipitsidwa koyamba, kuipitsidwa kwa zida za labotale, kuipitsidwa kwa aerosol, ndi zina zotero, kumafunika kugwiritsa ntchito RNase scavengers ndi RNase inhibitors.Kuwonongeka kwa aerosol ndikovuta kwambiri kupeza.Tangoganizani kuti labotale yanu ili ngati utsi, yokhala ndi ma nucleic acid osiyanasiyana atalendewera mumpweya.
NRT (No-Reverse Transcriptase), kuwongolera kopanda kulembedwa mobwerezabwereza, ndi RNA yosasinthika ngati kuwongolera koyipa, komwe ndikuwongolera zotsalira za gDNA.
Pochita mafotokozedwe a jini, kuchuluka kwa RNA kumadziwika pozindikira kuchuluka kwa cDNA pambuyo polemba m'mbuyo.Ngati pali zotsalira za gDNA pamene RNA yayeretsedwa, idzayambitsa zolakwika muzotsatira zoyesera, chifukwa zotsatira zenizeni zomwe zimapezeka ndi gDNA ndi cDNA.Pamlingo wophatikizika, osati cDNA yokha, gDNA iyenera kuchotsedwa kwathunthu pakuchotsa RNA.
MIQE (2)—chidziwitso chachitsanzo
Zomwe zimatchedwa zachitsanzo zikutanthauza kuti tikafalitsa nkhani yokhudza qPCR, tiyenera kufotokozera zachitsanzozo momveka bwino, zomwe ndi gawo lofunikira kwambiri pankhaniyi.Mofananamo, tikamakonza zitsanzo, tiyeneranso kuyang'anira ntchito zathu kuti tiwonetsetse kuti zitsanzozo ndizovomerezeka.
Kufotokozera kwa chitsanzocho ndi zotsatira chabe, ndipo tiyenera kumvetsera kwambiri zipangizo zomwe zimatengedwa panthawi yonse yoyesera.
Kusankha zipangizo zoyesera
Zitsanzo za magazi - sankhani magazi atsopano, osapitirira maola anayi.Zitsanzo za ma cell - sankhani kusonkhanitsa maselo atsopano mu nthawi ya kukula kwakukulu.Minofu Ya Zinyama-Sankhani minofu yatsopano, yomwe imakula mwamphamvu.Zomera Zomera - Sankhani minofu yatsopano, yachinyamata.
Muyenera kuti mwazindikira kuti pali mawu ofunikira m'mawu ochepa awa: mwatsopano .
Pazitsanzo zapamwambazi, zida zabwino kwambiri, zotsika mtengo, komanso zokhazikika pamsika ndi zida za Foregene, zomwe zimatha kuchotsa DNA ndi RNA mwachangu komanso mosavuta.
Animal Total RNA Isolation Kit
Plant Total RNA Isolation Kit Plus
Kusungirako zinthu zoyesera
Nthawi zambiri, sitimalimbikitsa kusunga zitsanzo, ngati ziloleza.Komabe, pali abwenzi ambiri omwe sangathe kuyesa mayeso atangotenga zitsanzo, ndipo ena amafunika kunyamula matanki amadzimadzi a nitrogen kupita nawo kumunda kuti akatengere zitsanzo.
Kwa mtundu uwu wa bwenzi wolimbikira ntchito, ndingangonena kuti simukumvetsetsa zogwiritsira ntchito reagent.Tsopano makampani ambiri ogwiritsira ntchito reagent amapanga ma reagents omwe amatha kusunga zitsanzo za RNA kutentha kwa firiji, ndipo mutha kusankha kuzigwiritsa ntchito.Njira yosungiramo wamba ndi kusungirako nayitrogeni wamadzimadzi, pogwiritsa ntchito thanki yaing'ono yamadzi ya nayitrogeni yomwe ndi yosavuta kunyamula.Mukabweretsanso chitsanzocho ku labotale, chisungeni mufiriji -80 ° C.
Pazoyeserera zokhudzana ndi RNA, mfundo ya mawu asanu ndi limodzi iyenera kutsatiridwa:kutentha kochepa, palibe ma enzymes,ndikudya .
Lingaliro la kutentha kochepa ndilosavuta kumvetsa;popanda ma enzyme, RNase ili paliponse padziko lapansi lomwe tikukhalamo (kupanda kutero mukadaphedwa ndi kachilombo ka HIV), ndiye momwe mungapewere RNase poyesa ndi lingaliro lofunika kwambiri;kudya,Palibe Kung Fu padziko lapansi komwe sikungathe kusweka, kuthamanga kokha sikungathe kusweka.
Choncho, tinganene kuti kufupikitsa nthawi yochotsa zinthu kumapangitsa kuti zidazo zikhale bwino.Chifukwa chiyani?Foregene's zida kutsindika liwiro, chifukwa amadziwa bwino.
PS: Atsikana ena amayesa mosamala kwambiri, koma sakhala abwino ngati slam dunk atatha zaka zingapo akugwira ntchito.Iwo amaona kuti Mulungu alibe chilungamo, amadandaula za ena, ndiponso amafunafuna moyo.Ndipotu sanamvetse.Sanateteze bwino RNA, ndipo wosewera mpira wa slam dunk anali wosavuta.Pamene anali kuchita kuyesera, ankaganiza kuti akamaliza slam dunk ndi katatu, kasanu ndi magawo awiri, koma iye anachita bwino.
Zindikirani: Pang'onopang'ono, mwayi wochuluka wa kuwukira kwa RNase.Kodi mungadziphunzitse bwanji kuti mukhale othamanga?Palibe njira, ingochita zambiri.
Pazoyeserera zosiyanasiyana ndi zitsanzo zosiyanasiyana, ndikofunikirabe kuwerenga mabuku ambiri ndikusankha njira yoyenera yosinthira.Pakusonkhanitsa ndi kusungirako zitsanzo, MIQE imafuna kuti ilembedwe bwino papepala, kuti owunikira awonenso kudalirika kwa pepalalo, komanso ndikwabwino kwa achinyamata odabwitsidwa kubwereza zomwe mwayesa.
Ngakhale kuyesa kwachilengedwe kumakhala kovuta, ndipamwamba kwambiri.Ngati simusamala, mutha kugwetsa dziko lapansi.Mwachitsanzo, kupanga SARS kukhala vuto lazachilengedwe, kapena kupanga mpunga wosakanizidwa kupulumutsa anthu mabiliyoni 1.3.Chithunzi chili m'munsichi ndi kuyesa mankhwala, muyenera kumvetsetsa momwe mumanyadira kafukufuku wanu pongoyang'ana maonekedwe ake ngati dick.Iwalani, musamudetse.
MIQE (3) - nucleic acid m'zigawo.
Kutulutsa kwa nucleic acid ndi chochitika chachikulu, ndipo kuyesa konse kwa biology ya maselo kumayamba ndikuchotsa nucleic acid.Choyamba, tiyeni titengere zomwe MIQE ili nazo pakuchotsa ma nucleic acid.
Kuyang'ana mawonekedwe awa, simungathe kukhala pamwamba.Mawonekedwewo ndi chiphunzitso.Kuti mukhale wophunzira wapamwamba, muyenera kufunsa chifukwa chake.Zofunikira pa tebulo ili ndi izi: Tsatiranichiyero, kukhulupirika, kusasinthasintha, ndi kuchuluka kwa RNA .
Gawo loyamba landondomeko kapena chida ndi sitepe ya nucleic acid m'zigawo.Ngati mugwiritsa ntchito makina opangira ma nucleic acid kuti muchotse (zambiri, chonde nditumizireni kuti mugule), muyenera kuwonetsa dzina lachitsanzo la chidacho.
Dzina la zida ndi
ndi zida zotani zomwe zidagwiritsidwa ntchito pazosintha, ndi ma reagents apadera ati omwe adawonjezedwa kapena ntchito zapadera zomwe zidachitika ziyenera kufotokozedwa momveka bwino kuti ena athe kubwereza kuyesa kwanu mosavuta.
Anthu ena amawonjezera ma reagents apadera pochotsa zitsanzo zapadera, poganiza kuti ichi ndi chida chawo chachinsinsi ndipo osauza ena.Pomwe amasunga chinsinsi, amatayanso mwayi wopanga nkhani yanu.Musakhale ochenjera, muyenera kukhala owona mtima kuposa dziko lakale la Zhang mu kafukufuku wa sayansi, ngati mukufuna kukhala wochenjera, nkhaniyi idzakupangani kukhala opusa.
ayenera kukumbukira chiwerengero cha mankhwala a zidamukayitanitsa zida ndikulemba nkhaniyo.Nthawi zambiri pamakhala manambala awiri pa zida: Nambala ya mphaka (nambala yazinthu, nambala yankhani), Loti - Nambala yachinthu (Yogwiritsidwa ntchito posonyeza gulu lomwe katunduyo adachokera).
Kuphatikiza apo, nambala ya CAS imagwiritsidwa ntchito nthawi zambiri poyitanitsa ma reagents a biochemical, ndipo ndidzayifalitsa palimodzi.Nambala ya CAS ndi nambala yoperekedwa ndi American Chemical Society ku mankhwala atsopano aliwonse.Nthawi zambiri, manambala atatu amalumikizidwa ndi mzere.Nambala ya CAS ya Rushui: 7732-18-5.Mankhwala nthawi zambiri amakhala ndi ma alias angapo, koma nambala ya CAS ndi yapadera.Mukayitanitsa mankhwala, mutha kuyang'ana nambala yake ya CAS poyamba.
Kufupi ndi kwathu, n’chifukwa chiyani tiyenera kufotokoza zinthu zimenezi momveka bwino?M'malo mwake, ndikuwunikanso mtundu wa RNA m'zigawo.Kugwiritsa ntchito zida ndi zida kumapangitsa kuchotsa kwa RNA kukhala kogwirizana.Kuchuluka kwa ma laboratories wamba sikuli kwakukulu, ndipo kumatha kupezeka ndi zida.
Tsatanetsatane wa chithandizo cha DNase kapena RNase
Nkhani yofunikira ya fluorescent quantitative PCR ndiyo kupewa kuipitsidwa kwa DNA, ndipo musayese ngati pali kuipitsidwa.Chifukwa chake, ndikofunikira kunena njira yomwe mudagwiritsa ntchito popanga DNA, kuti muwonetse kuti DNA muzoyeserera yachotsedwa kwathunthu.kuimiridwa ndi chithunzi cha schema.
Chithunzi cha RNA ndi DNA
Nthawi zambiri, njira yochotsera DNA ndikuchiza RNA ndi DNase pambuyo pochotsa.Komabe, izi ndi njira zakale.Zida zamalonda za RNA zowonjezera zatha kuchotsa DNA panthawi yochotsa popanda kuwonjezera DNase.Mwachitsanzo, zida zingapo zochokera ku Foregene.
Zindikirani: Kuchotsa DNA panthawi yochotsa RNA ndi lupanga lakuthwa konsekonse koopsa, lomwe lidzatalikitsa nthawi yogwira ntchito ya RNA ndikuwonjezera chiopsezo cha kuwonongeka kwa RNA.Kwenikweni, ndikugulitsa pakati pa zokolola za RNA ndi chiyero.
Kuonjezera apo, kuchuluka kwa DNase kuwonjezeredwa ku silika-based adsorption column ndi yochepa kwambiri, ndipo DNase yapamwamba iyenera kugwiritsidwa ntchito kuti ikwaniritse zotsatira zake.DNase yosakwanira siyingagayidwe mwachangu komanso kwathunthu.Uku ndi kuyesa kwaukadaulo wamalonda.Inde, pali amalonda odabwitsa kwambiri omwe amadzitamandira kuti DNA ikhoza kuchotsedwa popanda DNase.Zinganenedwe kuti aliyense amene amadzitamandira kuti DNA ikhoza kuchotsedwa kwathunthu popanda DNase ndi chiwonongeko.DNA ndi yokhazikika yazingwe ziwiri, ndipo siingathe kuchotsedwa mwa kungolankhula ndi kuseka.
Kuwunika kwa kuipitsidwa
Njira yowunikira: kuzindikira kwa electrophoresis, 1% agarose, 6V / cm, 15min, kutsitsa 1-3 ul
Nucleic acid kuchuluka kusanthula
Nthawi zambiri amayezedwa pogwiritsa ntchito UV spectrophotometer.Ndiroleni nditchule kaye tanthauzo la zinthu zitatu za OD260, OD280, ndi OD230.
·OD260nm: Ndiko kuyamwa kwa kutalika kwa nsonga yapamwamba kwambiri ya nucleic acid, ndipo mtengo wake wabwino kwambiri umachokera ku 0.1 mpaka 1.0.Ngati sichoncho, chepetsani kapena sungani kwambiri chitsanzocho kuti chikhale chofikira.
OD280nm: Ndiko kuyamwa kwakutali kwa nsonga yapamwamba kwambiri ya mayamwidwe a mapuloteni ndi zinthu za phenolic.
·OD230nm: Ndiko kuyamwa kwakutali kwa nsonga yapamwamba kwambiri yama carbohydrate.
Kenako, tiyeni tikambirane udindo wa aliyense chizindikiro.Kwa A260, itha kugwiritsidwa ntchito kuyeza zokolola za nucleic acid.Pamene OD260=1, dsDNA=50μg/ml, ssDNA=37μg/ml, RNA=40μg/ml.
Pa chiyero, tiyenera kuyang'ana mareyiti omwe timakonda kuwona: OD260/280 ndi OD260/230.
DNA Yoyera: OD260/280 pafupifupi yofanana ndi 1.8.Zikakhala zazikulu kuposa 1.9, zimasonyeza kuti pali kuwonongeka kwa RNA, ndipo pamene zili zosakwana 1.6, zimasonyeza kuti pali kuwonongeka kwa mapuloteni ndi phenol.
RNA Yoyera: 1.7
·OD260/230: Kaya ndi DNA kapena RNA, mtengo wake ndi 2.5.Zikakhala zosakwana 2.0, zimasonyeza kuti pali kuipitsidwa kwa shuga, mchere ndi zinthu zamoyo.
Kukhulupirika kwa RNA
Ndikofunikira kwambiri kuyeza kukhulupirika kwa RNA.Nthawi zambiri, pamafunika kuyesa gel osakaniza a RNA kuti muwone ngati kuwala pakati pa 28S ndi 18S RNA ndi ubale wapawiri.Pamene gulu lachitatu la 5S likuwonekera, zikutanthauza kuti RNA yayamba kunyozeka, kupatula zamoyo zopanda msana.
Deta ya RNA yowunika khalidwe: Kuphatikiza pa mayesero omwe ali pamwambawa, palinso mayesero ena apamwamba kwambiri a zida za RNA, monga kuyesa kwa umphumphu wa RQI wa Experion automatic electrophoresis system, yomwe imatha kuzindikira ngati RNA yawonongeka mosawoneka.
Mu kafukufuku wa sayansi, fulorosenti kuchuluka kwa PCR ndikuyerekeza pakati pa jini yomwe mukufuna ndi jini yolozera mkati.Choncho, pokonzekera kusungirako zitsanzo za RNA, kuchotsa RNA, ndi zina zotero, cholinga chachikulu ndikuwonetsetsa kukhulupirika kwa RNA.
Momwe umphumphu wa RNA umakhudzira kulinganiza pakati pa jini yowunikira ndi jini yolozera mkati imatha kumveka mosavuta kuchokera pachithunzi pansipa.Kuwonongeka kumabweretsa kusakwanira kwa jini, kaya ndi kusakwanira kwa jini yofotokozera zamkati kapena kusakwanira kwa jini yomwe mukufuna, zidzakhudza kwambiri deta.
Chiwonetsero cha jini chandamale ndi jini yolozera, sichiyenera kukhala chowona
Kuyesa koletsa (ngati mtengo wa CT umaponderezedwa pansi pa ndende yayikulu kapena yotsika kapena zina)
Kutengera chithunzichi mwachitsanzo, ma Ct ma curve asanu ndi awa.Kugawidwa kwa ma CT pakati pa ma curve ndi osagwirizana, ndipo ma Ct amachedwetsa pansi pazigawo zapamwamba komanso zochepa, zomwe zimakhala zolepheretsa PCR.
Mfundo yofunika: Pochotsa RNA, tiyenera kusiya malingaliro olakwika ndikukhazikitsa olondola.
Lingaliro lolakwika ndilakuti: Kuchotsa RNA kumangotsatira zokolola, poganiza kuti kuchulukitsa kwa RNA komwe kumapezeka, kumakhala bwinoko.M'malo mwake, tikapanga quantification, ngati kuchuluka kwa majini sikuli kwakukulu, sitifunikira RNA yambiri.Kuchuluka kwa RNA komwe mumatulutsa ndikokwanira.
Lingaliro lolondola ndi:Kutulutsa kwa RNA kuyenera kutsata chiyero, kukhulupirika komanso kusasinthika.Kuyera kumatha kuwonetsetsa kuti zolemba zotsatizanazi sizikuletsedwa ndipo deta sidzakhudzidwa ndi DNA.Kukhulupirika kumatsimikizira kutsatana kwa zomwe mukufuna komanso zolozera zamkati.Kukhazikika kumatsimikizira kutsitsa kwachitsanzo chokhazikika.
MIQE (4) - reverse transcript
Maganizo olakwika: kufunafuna kuchuluka kwa zitsanzo.
Lingaliro lolondola: Tsatirani kusasinthika (kukhazikika), mosasamala kanthu za kuchuluka kwa RNA yonyamulidwa, mphamvu yolembera mosinthana imakhalabe yokhazikika, kuwonetsetsa kuti kusiyana kwa cDNA kumatha kuwonetsadi kusiyana kwa mRNA.
Timalongosola ndondomekoyi ndi chithunzi chojambula:
Chithunzi chojambula chojambula bwino, musakhale owona
Choyamba, tiyenera kumvetsetsa kusiyana pakati pa ndondomeko yolembera mobwereza ndi ndondomeko ya PCR.PCR imadutsa njira zingapo zotenthetsera ndi zowotchera, ndipo chidutswa chandamale chimakula kwambiri;pamene kulembera kumbuyo kulibe ndondomekoyi, tikhoza kuganiza kuti kulembera kumbuyo kumakhaladi chimodzi ndi chimodzi Panthawi yobwerezabwereza, zidutswa zambiri za RNA.
monga pali atha kupeza zidutswa zambiri za cDNA Information, ziyenera kumveka tsopano, chifukwa zidutswa zazikulu ndi zazing'ono zalembedwa mosinthana, ndipo sizingatheke kuyang'ana pachidutswa chimodzi.Ndipo chifukwa kuchuluka kwa RNA ndi kochepa, kuchuluka kwa cDNA komwe kumapezeka nakonso kumakhala kochepa, mosiyana ndi PCR, yomwe imakhala ndi mphamvu yokulitsa, kotero ndizosatheka kuzizindikira.
Zotsatira za cDNA electrophoresis
Kachiwiri, momveka bwino, kumasulira mobwerezabwereza kumachitika kamodzi-kamodzi, koma palibe reverse transcriptase kuchokera ku kampani iliyonse yomwe ingakwaniritse izi.Kwenikweni, kuthekera kwa zolemba zambiri zosinthira kumangoyendayenda pakati pa 30-50%.Ngati ndi choncho, tikadakonda kukhala ndi zolembera zosinthika mokhazikika, zomwe tikufuna kuwona pachithunzichi: 3 RNAs imapeza ma cDNA 2, ma RNA 6 amapeza ma cDNA 4, kotero mosasamala kanthu za kuchuluka kwa zitsanzo zomwe zapakidwa , zolembera zosinthika zimakhala zokhazikika.Sitikufuna kuwona momwe zolembera zosinthira ndizosakhazikika komanso kusasunthika kwakukulu kumaletsedwa.
Ndiye mungatsimikize bwanji ngati zomasulirazo ndizokhazikika?Njirayi ndi yophweka, muyenera kungoyesa kuyerekezera: imodzi ndikusintha kulembera mu cDNA pambuyo pa kuwirikiza kawiri kwa RNA, ndipo ina ndiyo kupanga kuwirikiza kawiri mutatha kubwereza ku cDNA, kenako qPCR kuti muwone malo otsetsereka.Monga wophunzira wapamwamba, muyenera kumvetsetsa mumasekondi.Monga momwe zilili pansipa:
Kuchepetsa kwa RNA ndi cDNA kuti muwone ngati kugwira ntchito kwa reverse transcription ndikokhazikika
Reverse transcriptase ndi kit
Kodi PCR yabwino kwambiri ya fulorosenti ingakhale bwanji ndi reverse transcriptase ndi zida.Reverse transcriptase imagawidwa m'mitundu iwiri malinga ndi gwero, AMV kapenaM-MLV, ndipo machitidwe awo ndi ofanana ndi omwe akuwonetsedwa patebulo.
RNase H ntchito
RNase H ndi Ribonuclease H, dzina lachi China ndi ribonuclease H, lomwe ndi endoribonuclease yomwe imatha kutulutsa RNA mu DNA-RNA hybrid chain.RNase H sangathe hydrolyze zomangira phosphodiester mu DNA ya nsonga imodzi kapena iwiri kapena RNA, ndiko kuti, sangathe kukumba DNA kapena RNA yamtundu umodzi kapena iwiri.Amagwiritsidwa ntchito popanga chingwe chachiwiri cha cDNA.
Ndi chinthu chachilendo.Tikunena kuti reverse transcriptase ili ndi ntchito ya RNase H, osati kuti reverse transcriptase ili ndi RNase H, ndipo sizingatheke kulekanitsa RNase H kuchokera ku reverse transcriptase, mwina chifukwa cha kugwirizana kwa magulu ena mu reverse transcriptase Ntchitoyi imayambitsidwa ndi reverse transcriptase.
Chifukwa chake, mosasamala kanthu kuti AMV imagwira ntchito bwino bwanji, ntchito yake ya RNase H imachepetsa zokolola za cDNA.Zachidziwikire, opanga ma reagent akukonza zinthu zawo nthawi zonse kuti athetse ntchito ya RNase H mu reverse transcriptase momwe angathere kuti awonjezere zokolola za cDNA.
Annealing kutentha
Mapangidwe achiwiri a RNA pa kutentha kosiyana
Onani chithunzi pamwambapa cha mawonekedwe achiwiri a RNA pa kutentha kosiyana, ndipo gwiritsani ntchito chida cha pa intaneti cha mFold kuti mudziwe mawonekedwe achiwiri a chidutswa chandamale pansi pa kutentha kwapadera ndi mikhalidwe yamchere.Pa 55 ° C, dongosolo lachiwiri la RNA likadali lovuta kwambiri, reverse transcriptase silingagwire ntchito, ndipo mawonekedwe achiwiri sangathe kuthetsedwa mpaka 65 ° C, pamene kutentha kwakukulu kwa AMV ndi M-MLV kumakhala kotsika kwambiri kuposa kutentha uku.
Zoyenera kuchita?Kapangidwe kachiwiri ndikuphatikizana kophatikizana kwa template yokha, komwe kumabweretsa mpikisano wamphamvu pakati pa primer ndi reverse transcriptase ndi template, zomwe zimapangitsa kuti pakhale zovuta zingapo monga E otsika komanso kusabwereza kobwerezabwereza.
Zoyenera kuchita?Ingowonjezerani kutentha kwa annealing momwe mungathere.
Opanga ma reagent ambiri akuwongolera reverse transcriptase yawo kudzera mu genetic engineering.Ena amawonjezera kutentha, monga Jifan ndi Aidelai, ndipo ena amachotsa gulu logwira ntchito la RNase H enzyme kuti apititse patsogolo kuyanjana pakati pa enzyme ndi template ya RNA.Kugwirizana kwakukulu kumatha kufinyira mawonekedwe achiwiri mopikisana ndikuwerenga bwino, komanso kumapangitsanso bwino kumasulira mobwerezabwereza.
Mfundo yofunika kwambiri: Reverse transcript ndiyofunika kwambiri kuti titsatire kusasinthasintha kwa reverse transcript performance (ma enzyme sayenera kukhala opambana komanso okhazikika), osati kuchuluka kwa zitsanzo zomwe zanyamulidwa, ngati sizili PCR yaikulu kwambiri ya fulorosenti, sizingatheke nkomwe.Ma cDNA ambiri.
Opanga osiyanasiyana apanganso zoyesayesa zina pofunafuna kusasinthika.Mwachitsanzo, makampani ambiri tsopano apanga zolembera zosinthika ngati zida zogulitsa, zomwe ndi chisankho chabwino.
Mwachitsanzo, zida za Foregene's RT Easy Series:
RT Easy I (Master premix for first strand cDNA synthesis kit)
MIQE (5) - chidziwitso cha jini chandamale
Chithunzi pamwambapa chikufotokoza
1. Ngati jini iyi ndi yothandiza poyesera mobwerezabwereza ikhoza kutsimikiziridwa ndi kuyesa mobwerezabwereza.
2. Gene ID, mukudziwa.
3. Utali wa jini, utali wonse wa jini yomwe mukufunayo ndiye palibe vuto.Mukamapanga zoyambira, onetsetsani kuti kutalika kwa amplicon kuli pakati pa 80-200bp kuti muwonetsetse kukulitsa bwino.
4. Tsatanetsatane wa kufananiza kwa Blast, jini yomwe mukufuna ikuyenera kufananizidwa mu genebank kuti mupewe kukulitsa kosakhazikika.
5. Kukhalapo kwa pseudogenes.A pseudogene ndi mndandanda wa DNA wofanana ndi jini wamba koma imataya ntchito yake yanthawi zonse.Nthawi zambiri amapezeka m'gulu lamitundu yambiri ya eukaryotes.Nthawi zambiri imayimiridwa ndi ψ.Ndikope ya DNA yosagwira ntchito mu genome yomwe ili yofanana kwambiri ndi kalembedwe ka jini., kaŵirikaŵiri sizimalembedwa, ndipo zilibe tanthauzo lenileni la thupi.
6. Udindo wa zoyambira poyerekeza ndi ma exons ndi introns.M'zaka zoyambirira, tikamathetsa vuto la kuipitsidwa kwa DNA, nthawi zambiri tinkasamala za malo oyambira, ma exons, ndi ma introns, ndipo nthawi zambiri tinkaganiza zopanga zoyambira pama introns kuti tipewe kukulitsa kwa DNA.Chonde onani chithunzi pansipa: wakuda amayimira ma introns, mabuluu osiyanasiyana amayimira ma exons, pinki amayimira zoyambira wamba, ndipo zofiira zowala zimayimira zoyambira zoyambira.
Schematic, osati zoona
Izi zikuwoneka ngati dongosolo labwino bwanji, koma kwenikweni, nthawi zambiri, ma trans-intron primers sakhala amatsenga monga momwe amaganizira, ndipo amayambitsanso kukulitsa kosagwirizana.Choncho njira yabwino yopewera kuipitsidwa kwa DNA ndiyo kuchotsa DNA kotheratu.
7. Kuneneratu za kusintha.Pogwiritsa ntchito chitsanzo ichi kachiwiri, gwiritsani ntchito chida cha mFold pa intaneti kuti mudziwe mawonekedwe achiwiri a chidutswa chandamale pa kutentha kwapadera ndi mchere.
Mapangidwe achiwiri a RNA pa kutentha kosiyana
Mapangidwe achiwiri ndi kuphatikizika kowonjezera kwa template komweko, komwe kudzadzetsa mpikisano wamphamvu pakati pa oyambira ndi ma template, ndipo mwayi womangirira woyambira umakhala wocheperako, zomwe zimapangitsa kuti pakhale zovuta zingapo monga kutsika kwa E komanso kubwereza koyipa.Kupyolera mu kulosera kwa mapulogalamu, ngati palibe vuto lachiwiri la mapangidwe, zingakhale zabwino.Ngati alipo, nkhani yathu yotsatira idzafotokoza mwatsatanetsatane mmene tingathetsere vutoli.
MIQE (6)—qPCR Oligonucleotides
Kwa PCR yochulukirachulukira, chinthu choyamba chomwe mumalimbana nacho tsiku lililonse ndikuchotsa kwa RNA, ndipo chachiwiri chingakhale kapangidwe kake.
Choyamba, timayang'anabe malamulo okhudza mapangidwe oyambira malinga ndi mndandanda wa MIQE.Ndizosavuta kuti scumbags akhoza kuseka, ndipo tikhoza kumaliza mu chiganizo chimodzi: fufuzani ndondomeko ndi malo a kafukufuku woyamba ndi njira yosinthira.Kwa njira yoyeretsera zoyambira, kaphatikizidwe koyambira ndi kotchipa kwambiri pakadali pano, qPCR ndiyoyenera PAGE komanso njira zoyeretsera, ndipo chidziwitso cha chida chophatikizira sichifunikira.Anthu ambiri akhala akuchita zoyambira kwazaka zambiri ndipo sakudziwa kuti synthesizer ndi ABI3900.
Pankhani ya mfundo za kapangidwe koyambirira, simuyenera kuziloweza mwa rote, chifukwa mapulogalamu ambiri oyambira kapena zida zapaintaneti zimatha kuthana ndi mavutowa (chida chovomerezeka chapaintaneti primer3.ut.ee/), ndipo 99.999% ya mapangidwe oyambira samachitidwa pamanja.
Ingoyang'anani mfundo zotsatirazi pambuyo popanga zoyambira:
1. Zojambula zoyambira pafupi ndi 3 'mapeto: Pankhani yogwiritsira ntchito oligo dT primers kwa cDNA yoyamba-strand synthesis, poganizira reverse transcription efficiently and RNA integrity, primers yopangidwira iyenera kupangidwa pafupi ndi 3' mapeto kuti apititse patsogolo mphamvu zowonjezera.Gwiritsani ntchito chithunzi kuti mufotokoze motere (palibe njira yomvetsetsa izi):
Chifukwa chiyani zoyambira ziyenera kupangidwa pafupi ndi mapeto a 3, siziyenera kukhala zoona
2. Mtengo wa TM: Mtengo wa Tm uli pa 55-65 ° C (chifukwa ntchito ya exonuclease ndipamwamba kwambiri pa 60 ° C), ndipo GC ili pa 40% -60%.
3. KUBULA: Kuti mupewe kukulitsa kwamtundu wina, Blast iyenera kugwiritsidwa ntchito potsimikiziranso.
MIQE(7)—qPCR ndondomeko
1. qPCR zida
Malinga ndi zofunikira za MIQE, tiyenera kufotokoza momveka bwino momwe zinthu zilili m'nkhaniyo, kuphatikizapo kasinthidwe ka PCR reaction system, zida zomwe zimagwiritsidwa ntchito, ndani wopanga, momwe zimakhalira zazikulu, kaya njira ya utoto kapena njira yofufuzira ikugwiritsidwa ntchito, makonzedwe a pulogalamu ya PCR.Madalaivala akale adzapeza kuti malinga ngati zida zasankhidwa, zomwe zili pamwambazi zimatsimikiziridwa.
Pakali pano, kupanga ndi kupanga fulorosenti kachulukidwe PCR zida ndi luso okhwima kwambiri.Malingana ngati simusankha opanga zoyipa kwambiri, mwayi wamavuto siwokwera, komabe tikufuna kugawana nanu mfundo zingapo:
Enzyme ya Taq yotentha:Gawo lofunika kwambiri la PCR ndi enzyme ya Taq yotentha.Ma enzymes oyambira otentha pamsika nthawi zambiri amagawidwa m'mitundu iwiri, imodzi ndi enzyme yosinthidwa ndi mankhwala (mutha kuiganizira ngati kuyika kwa parafini), ndipo inayo ndi Kodi enzyme yoyambira yotentha yosintha ma antibody (yomanga ma antigen-antibody).Kusintha kwa Chemical ndi njira yoyamba yoyambira ma enzymes.Pamene kutentha kwina kwafika, enzyme imamasula ntchito yake.The antibody-modified hot-start enzyme imagwiritsa ntchito njira zachilengedwe kuletsa ntchito ya enzyme.Kutentha kwina kukafika, antibody imasinthidwa ndikusinthidwa ngati puloteni, ndipo ntchito ya enzyme imayamba kugwira ntchito.
Komabe, kugwiritsa ntchito izi ndi chiyani?Izi zili choncho, ntchito yotulutsa ma enzymes osinthidwa ndi ma antibody imathamanga kwambiri kuposa ma enzymes osinthidwa ndi mankhwala, kotero potengera kukhudzidwa, ma enzyme osinthidwa ndi ma antibodies ali ndi mwayi pang'ono, kotero kuti palibe ma enzymes osinthidwa ndi mankhwala pamsika.Ngati alipo, ndiye kuti luso la wopanga uyu lidakalipobe mu nthawi ya zaka chikwi.
Magnesium ion concentration:Magnesium ion concentration ndi yofunika kwambiri pakuchita kwa PCR.Kuphatikizika kwa magnesium ion kumathandizira kutulutsidwa kwa ntchito ya Taq enzyme.Ngati ndende ndi yotsika kwambiri, ntchito ya enzyme idzachepetsedwa kwambiri;Ngati ndendeyo ili yochuluka kwambiri, kukulitsa kwa enzyme-catalyzed non-specific amplification kumawonjezeka.Kuphatikizika kwa ayoni a magnesium kudzakhudzanso kuyambika kwa zoyambira, kutentha kwa template ndi zinthu za PCR, potero kukhudza zokolola za zidutswa zokulitsa.Kuchuluka kwa ayoni a magnesium nthawi zambiri kumayendetsedwa pa 25mM.Zachidziwikire, kuti mukhale ndi zida zabwino, kuchuluka kwa ayoni a magnesium kuyenera kuyendetsedwa bwino.Amalonda ena amawonjezera magnesium ion chelating agent ku reagent, yomwe imatha kukwaniritsa kusintha kwa ndende ya magnesium ion.
Kuchuluka kwa utoto wa fluorescent:Utoto wa fluorescent, womwe ndi SYBR Green womwe timakonda kugwiritsa ntchito, makamaka umapanga fluorescence pomangirira ku nsonga yaying'ono ya DNA yamitundu iwiri, chifukwa kumangirira utoto ku DNA yamitundu iwiri sikuli yeniyeni, ndiko kunena kuti bola ngati DNA yazingwe ziwiri ikuphatikizidwa ndi iyo, DNA imaphatikiziridwa ndi ma templates, ma fluores ndi ma templates. kupanga chizindikiro chakumbuyo.
PS: Chifukwa cha mawonekedwe ake osamva kuwala, zogulitsa pamsika nthawi zambiri zimayikidwa mu machubu a bulauni opaque centrifuge (monga momwe chithunzi chili pansipa).Komabe, izi zidzakumana ndi vuto.Ndizovuta kuwona ngati madziwo amayamwa poyesa.Pachifukwa ichi, Qingke ndiyomwe imakonda kwambiri ogwiritsa ntchito (monga momwe tawonetsera pachithunzichi), ndipo chubu chowonekera chimayikidwa mu thumba la malata opaque.Kenako ikani m'thumba la malata, poganizira za kupeŵa kuwala ndi zitsanzo.Muyenera kusankha nambala yoyenera yamalonda.TSE204 ndi moyo wotsika mtengo kwambiri, zomwe zimandipangitsa kufuna kubzala udzu.
Kuchuluka kwa utoto wa fulorosenti ndikofunikira kwambiri.Ngati ndendeyo ili yotsika kwambiri, mapindikidwe amplification sangakwere pambuyo pake ndipo siangwiro;ngati ndendeyo ndi yokwera kwambiri, imayambitsa kusokoneza kwa phokoso.Popeza kuchuluka kwa fulorosenti ya PCR makamaka kumadalira mtengo wa CT, ngati mtundu wa utoto wa fulorosenti sunasinthidwe bwino, malo otsika ndi abwino kuposa okwera.Zoonadi, mtundu woyenera wa utoto ndi wabwino kwambiri.
Mtengo ROX: Utoto wa ROX umagwiritsidwa ntchito kuwongolera zolakwika zamtundu wa fluorescence bwino.Opanga zida zina amafuna kuwongolera, pomwe ena satero.Mwachitsanzo, kugwiritsa ntchito chida chokulitsa cha Thermo Fisher Scientific's Real Time PCR nthawi zambiri chimafuna kuwongolera, kuphatikiza 7300, 7500, 7500Fast, StepOnePlus, ndi zina zambiri.
Foregene's qPCR Mix ilinso ndi utoto wa ROX, womwe ndi wosavuta kugwiritsa ntchito mitundu yosiyanasiyana.
Chithandizo chochepa cha hydrogen bond: Chithandizo cha ma bond ofooka a haidrojeni ndi nkhani yaukadaulo.Palibe chomwe chawerengapo zolemba zamakiti ambiri, koma palibe m'modzi mwa iwo amene adatchulapo mutuwu.Ndipotu n’kofunika kwambiri.Kuphatikiza zapansi makamaka zimadalira mphamvu ya hydrogen zomangira.Zomangira zamphamvu za haidrojeni ndizokulitsa bwino, ndipo zomangira zofooka za haidrojeni zimatsogolera kukulitsa kosagwirizana.Ngati zomangira zofooka za haidrojeni sizingathetsedwe bwino, kukulitsa kosakhazikika sikungapewedwe.Pankhani ya wolemba, ndi makampani ochepa okha omwe awona vutoli.Mukagula zida, mutha kunena ngati mwaganizirapo yankho pankhaniyi pa zida zomwe mukufuna kusankha.
Mphamvu yamphamvu: Dongosolo la 20-50ul limagwiritsidwa ntchito kwambiri, ndipo ma voliyumu ang'onoang'ono amatha kuyambitsa zolakwika.Nthawi zambiri, malangizo a zida amalimbikitsa kugwiritsa ntchito ma voliyumu a PCR.Musakhale anzeru ndikugwiritsa ntchito ma voliyumu ang'onoang'ono kuti musunge ndalama.cholinga cha.Voliyumu yomwe amalonda amalimbikitsa yayesedwadi, ndipo mwina sangathe kuthetsa vuto la zolakwika zomwe zimayambitsidwa ndi mabuku ang'onoang'ono.
2. Wopanga ndi nambala yankhani ya mbale ya chubu
Aliyense amadziwa mfundo ya fulorosenti kachulukidwe PCR.Kutolere fluorescence makamaka ikuchitika kudzera PCR chubu zisoti.Posankha PCR consumables, tcherani khutu ku mfundo ziwiri: uthenga kufala kuwala ndi oyenera chida.Nthawi zambiri, matabwa ndi machubu amtundu wodziwika bwino ndi abwino, koma muyenera kusankha mosamala potengera kusintha, apo ayi simungathe kugwiritsa ntchito chidacho.
4. Chidziwitso chapamwamba
MIQE (8)—qPCR kutsimikizira
Ichi ndiye chofunikira kwambiri cha qPCR!Ngwazi zambiri zagwera mumchenga pano.Inde, ndizothekanso kuti muli ndi mwayi ndipo majini omwe mudaphunzira ndi osavuta, kotero mudayandama kuphanga la ayezi motsatira mphepo.Zambiri zotsimikizira za qPCR cholinga chake ndikuyesa kudalirika kwa datayo.Timalemba zofunikira zotsimikizira motere:
1.Kuyesa kwapadera
Chidziwitso cha kukula kwa jini chandamale chimayesedwa poyang'ana ngati chithunzi cha electrophoresis ndi gulu limodzi;kutsimikizira kutsata;kusungunuka kokhotakhota kuti muwone ngati mapu apamwamba ndi amodzi;kutsimikizira chimbudzi cha enzyme ndi njira zina.
Apa, timayang'ana kwambiri pa tamasanthula kukulitsa kosakhazikika pogwiritsa ntchito njira yosungunulira.Nthawi zambiri, tikapanga zoyambira, kukula kwa chidutswacho kumafunika kukhala pakati pa 80-200bp, zomwe zimapangitsa kutentha kwazinthu za PCR kukhala 80-85 °C.Chifukwa chake, ngati pali nsonga zosiyanasiyana, payenera kukhala zinthu zina zokulitsa zomwe sizili zenizeni;ngati chiwombankhanga chikuwoneka pansi pa 80 ° C, nthawi zambiri chimatengedwa kuti ndi chiyambi cha dimer;ngati chiwombankhangacho chikuwoneka pamwamba pa 85 ° C, nthawi zambiri chimatengedwa kuti ndi DNA kuipitsidwa kapena kukulitsa Kwachidule kwa zidutswa zazikulu.
Chidziwitso: Nthawi zina pamakhala nsonga imodzi yokha pa 80°C.Panthawi imeneyi, mfundo iyi iyenera kutsatiridwa.Zikuoneka kuti zotsatira zake zonse ndi ma dimers oyambira.
Mpiringidzo wosungunuka wamba (chinsonga chimodzi chokha chopanda kukulitsa kwenikweni)
Mpendero wosungunuka wovuta (kukulitsa kosaneneka kwa nsonga zonyenga)
【Kusanthula nkhani】
Pali nsonga yayikulu, koma primer dimer ndi yayikulu
Mphepete mwa nsonga imodzi yosungunuka m'chithunzi chomwe chili pansipa chikhoza kunyenga maso anu mosavuta, kuganiza kuti ndi kuyesa koyenera, koma zotsatira zake ndizolakwika.Panthawi imeneyi, tiyenera kuyang'ana kutentha kusungunuka.Kutentha kwakukulu kumakhala pansi pa 80 ° C, komwe kumakhala primer-dimer.
Palibe chidutswa chandamale, ma dimers onse oyambira
Apa mchimwene wanga sangayime.Chithunzi chomwe chili pansipa ndi chithunzi chojambulidwa ndi foni yam'manja yotumizidwa kwa ine ndi scumbag.Ma reagents omwe amagwiritsidwa ntchito ndi onse omwe amagwiritsidwa ntchito kwambiri pamakampani.Anasintha kuchoka pamtundu wa T-prefix kupita ku mtundu wina wa T-prefix.Ndikuganiza kuti mwalingalira kale.Wonyansayo anandilirira kuti: “Chitsulo chimene chagwiritsidwa ntchito pachithunzi choyambacho ndi chabwino kwambiri, ndipo pachimake n’chachimodzi.Pambuyo pake, mutatha kugwiritsa ntchito reagent yomwe mudalimbikitsa, imakhala ngati chithunzi chachiwiri, chokhala ndi nsonga zosakanikirana.Mwandimvetsa chisoni.“
Kulekanitsa ma graph awiriwo.Poyamba, wina ali ndi nsonga imodzi, ndipo winayo ali ndi nsonga ziwiri.Zamkhutu, nsonga imodzi ndiyabwino.Kodi izo nzoona?
Choyipa kwambiri kuposa Dou E, ngati ndiyika zithunzi ziwiri pa chithunzi pansipa, mudzamvetsetsa nthawi yomweyo.M'malo mwake, timapuwala mosavuta ndi chithunzi chamtunduwu.Pambuyo pofufuza mosamala, tapeza kuti: nsonga ya chiwerengero choyamba ndi 75 ° C, yomwe imakhala yoyambira kwambiri;pachimake cha chiwerengero chachiwiri chikuwoneka pa 75 ° C ndi 82 ° C, osachepera pali Chogulitsacho chikuwoneka.
Zithunzi za ndemanga zochokera kwa ophunzira
Chifukwa chake vuto lalikulu si vuto la ma reagents, koma vuto la mapangidwe oyambira.Panthawi imodzimodziyo, zimatsimikiziranso kuti zizindikiro zina zazikulu siziri zachitsulo, komanso zimatsimikizira zomwe mchimwene wanga adanena kale: Si mtundu wa reagent umene umathandizira nkhani yanu.Ndi nkhani yanu yomwe idathandizira mtundu wa reagents.Tangoganizani, ngati scumbag sichinasinthe ma reagents, deta yolakwika idzatumizidwa ku magazini, ndipo zomwe zingachitike zingakhale zomvetsa chisoni.
2. Ct mtengo wa kulamulira opanda kanthu
Osalongosola, ngati kuwongolera kopanda kanthu kuli ndi mtengo wa Ct, sikuyipitsa?Komabe, mukufunikirabe kumvetsetsa kuti ndi chiwongolero chopanda kanthu chomwe chili ndi mtengo wa Ct.Ngati ndi NTC, zikutanthauza kuti pali DNA yakunja monga kuipitsidwa kwa reagent.Ngati ndi NRT, zikutanthauza kuti RNA yotengedwa ili ndi kuipitsidwa kwa DNA.
3. Mpiringidzo wokhazikika
Kuphatikizapo otsetsereka ndi formula yowerengera, mphamvu ya PCR imatha kuwerengedwa kudzera mu formula.Kuyesera koyenera kumafuna kutsetsereka kwa piritsi lokhazikika kuti muyandikire 3.32, ndi R² kuti muyandikire 0.9999.
4. Linear dynamic range
Zomwe zimasinthasintha zimasiyanasiyana.Malinga ndi template yomwe imagwiritsidwa ntchito popanga ma curve okhazikika, magawo osinthika akuyenera kukhala ndi ma concentration gradients osachepera 5, ndikulabadira kusintha kwa Ct pamiyezo yayikulu komanso ma gradient otsika.
5. Kuzindikira kulondola
Kusintha kwa zotsatira za qPCR, ndiko kuti, kusabwerezabwereza, ndiko kuti, kusalondola bwino, kumayambitsidwa ndi zinthu zambiri, kuphatikizapo kutentha, kukhazikika, ndi ntchito.Kulondola kwa qPCR nthawi zambiri kumakhala kosavuta kulamulirika pamene nambala yokopera imachepa.Moyenera mkati mwazoyeserera, kusiyanasiyana kwaukadaulo kumeneku kuyenera kukhala kosiyana ndi kusinthika kwachilengedwe, ndipo zofananira zachilengedwe zimatha kuthana mwachindunji ndi kusiyana kwa ziwerengero za zotsatira za qPCR pakati pamagulu kapena chithandizo.Makamaka pazowunikira zowunikira, kulondola kwapakati-kubwereza (kubwereza) pamasamba ndi ogwiritsa ntchito kuyenera kufotokozedwa.
6. Kuzindikira bwino ndi LOD (mu multiplex qPCR)
LOD ndiye ndende yotsika kwambiri ya 95% ya zitsanzo zabwino zomwe zapezeka.Mwanjira ina, kuchuluka kwa LOD komwe kuli mkati mwazotsatira zamtundu wa chandamale sikuyenera kupitilira 5% ya zomwe zidalephera.Pochita kafukufuku wa multiplex qPCR, makamaka kuti azindikire panthawi imodzi ya kusintha kwa mfundo kapena ma polymorphisms, multiplex qPCR ikuyenera kupereka umboni wosonyeza kuti kulondola kwa zidutswa zamagulu ambiri sikusokonezedwa mu chubu chomwecho, kufufuza kangapo ndi kuzindikira chubu limodzi Kuchita bwino ndi LOD ziyenera kukhala zofanana.Makamaka pamene chibadwa chandandandandanda chachikulu ndi chibadwa chandamale otsika kachulukidwe jini akukulitsidwa panthawi imodzi, vutoli ayenera kulabadira.
Mavuto ndi zothetseraNthawi zambiri, mavuto omwe nthawi zambiri amakumana nawo pakuwongolera qPCR amayang'ana mbali izi:
·kukulitsa mosadziwika bwino
· Kusankha kovuta kwa ndende ya primer ndi vuto ndi ma primer-dimers
•Kutentha kwa nthiti sikulondola
·Mapangidwe achiwiri amakhudza kukulitsa bwino
kukulitsa mosatchulika
kukulitsa kosakhazikikazimachitika , nthawi zambiri zimaganiziridwa ngati mapangidwe oyambira si abwino, koma ngati simukufulumira kusintha zoyambira, mutha kuyesa njira zotsatirazi poyamba (mfundoyi imaphatikizidwanso):
Onjezani kutentha kwa anneal - yesetsani kupanga ma bond ofooka a haidrojeni kuti asathe kusamalira;
•Kufupikitsa nthawi yofupikitsa ndi kutalikitsa - kuchepetsa mwayi wa ma bond ofooka a haidrojeni;
-Kuchepetsa kuchulukirachulukira - kuchepetsa mwayi wokhala ndi zoyambira zocheperako komanso madera omwe sakufuna;
Low amplification mphamvu
Zomwe zimatsutsana ndi kukulitsa kosatchulika - kukulitsa luso lochepa, komanso njira zothanirana ndi kutsika kwamphamvu ndizosiyana:
- Kutalikitsa nthawi yophukira ndi kukulitsa;
·Sinthani kukhala masitepe atatu PCR ndikuchepetsa kutentha kwa annealing;
· Kuonjezera ndende yoyambira;
Ps: Ophunzira ambiri omaliza maphunziro obadwa m'zaka za m'ma 90 safuna kuphunzira momwe angasinthire zoyeserera, ndipo akuyembekeza kuti zida zitha kuthetsa vutolo (ngati mukufuna kupita ku kampani ya reagent kukachita kafukufuku ndi chitukuko mukamaliza maphunziro), kwenikweni, opanga ma reagent nawonso amaganiza motere, ndikhulupilira kuti ndi zopusa Itha kugwiritsidwa ntchito mukayipeza, kotero opanga ma reagent athana ndi zovuta zambiri pakuyesa kulephera, kuphatikiza kulephera, kuphatikiza kulephera kulephera. H-bond mayamwidwe zinthu.Kuti athetse vutoli mosavuta, opusa amayenerabe kuwerenga kuyambika kwa kampani ya reagent kuti awone ngati pali chinthu chomwe chimatenga zomangira zofooka za haidrojeni.
Kusankha kovuta kwa ndende yoyambira komanso vuto ndi ma primer-dimers
Njira 1: Nthawi zambiri, malangizo a zida za qPCR amalimbikitsa machitidwe ndikulimbikitsanso zoyambira.
Njira 2: Kuthetsa vuto pokhazikitsa primer concentration gradient.Chithunzi chomwe chili pansipa chabedwa ku kampani kuti chiwonetsedwe.Chithunzi chomwe chili pansipa chikuwonetsa zotsatira za kuchuluka kwa ma fluorescence opangidwa ndi ma primer concentration gradients (100nM, 250nM, 500nM) ndi ma tempuleti anayi (0.1ng, 1ng, 10ng, 100ng).Mtengo wa Ct wa zotsatira zoyeserera umakonzedwa motere:
Kusankha Koyikirako Koyambira Gwirizanitsani zoyambira zilizonse kukhala mzere motere:
Kusankha koyambira koyambira ndi kodziwikiratu, kulumikizana kwa mzere wa ndende ya 100nM ndi 250nM ndikwabwinoko, ndipo kulumikizana kwa mzere wa ndende ya 500nM ndikocheperako.Mu 100nM ndi 250nM, mtengo wa Ct wa 250nM ndi wawung'ono, kotero kuti mulingo woyenera kwambiri wa primer ndi 250nM.Nthawi zambiri ma primer-dimers owopsa amatha kuwoneka pamapindikira osungunuka.Nanga bwanji ngati zoyambira zomwe zidapangidwa sizingapewe ma primer-dimers?
Njira 3: Chepetsani kuchuluka kwa zoyambira ndikuwonjezera kutentha kwa annealing (palibe chifukwa chofotokozera).
Mphamvu yamphamvu ya kutentha kwa annealing ndi 60 ° C.Ngati simuli otsimikiza, mungasankhe bwanji kutentha kwa annealing?Yankho ndilofanana ndi kusankha koyambira koyambira -mayeso a gradient.Tengani chithunzi kuchokera ku kampani ya Bio-rad kuti muwonetse vutoli.Pakukulitsa kwachidutswa chandamale chandamale, ikani ma gradients asanu ndi atatu, chilichonse chili ndi kubwereza katatu, ndipo pamapindikira okulitsa ndi motere:
kusankha kutentha kwa annealing:
· 70 ° C, 69 ° C - Kwenikweni, zoyambira sizingaphatikizidwe, kotero palibe kukulitsa.
· 67.3 ° C - Pali kukulitsa pang'ono poyambira, ndipo mtengo wa Ct ndi waukulu.
·64.5°C—— Mtengo wa Ct umachepa.
· Pa 60.7 ° C, 58.0 ° C, 56.2 ° C, ndi 55.0 ° C, mfundo za Ct makamaka zinkakhala zokhazikika, koma zomaliza za fluorescence zinali zosiyana.
Kodi kusankha?Mfundo Yofunika: Mfundo yoyamba ndi mtengo wapamwamba wa Ct.Pamtengo womwewo wa Ct, sankhani kutentha kopitilira muyeso kuti mupewe kuchepetsedwa ndi kukulitsa kosagwirizana kwenikweni.Ngakhale pali mtengo wapamwamba wa fluorescence pa 55 ° C, pangakhale ma dimers kapena osadziwika bwino momwemo.
Koma ngati muli anzeru monga inu, mudzaganiza motsimikiza: Kunena momveka bwino, ngati PCR ikuchita mwachindunji, malinga ngati ndondomeko yoyambira ikupitirira zofunikira zochepa, mfundo zapamwamba ndi zochepa siziyenera kukhala ndi zotsatira, monga utoto wa fulorosenti ndi dNTPs.Zowonadi, bola ngati kutentha kwa annealing kukhathamiritsidwa bwino, zotsatira za ndende ya primer pamtengo wa Ct zimachepetsedwa.
Kutentha kwa annealing kumakonzedwa bwino, ndipo zotsatira za primer concentration pa CT zidzachepetsedwa
Mapangidwe achiwiri amakhudza kukulitsa bwino
Tiyeni tijambule chithunzi kuchokera ku Bio-rad kuti tiwonetse vutoli.Imapanganso mawonekedwe a kutentha kuti akulitse jini yokhala ndi mawonekedwe achiwiri.
Mapangidwe achiwiri akuwonekera
Zitha kuwoneka kuti pamene kutentha kwa kutentha kumachepa, mankhwala amayamba kuonekera ndipo mtengo wa Ct umapita patsogolo, kufika pamtengo wocheperapo pa 60.7 ° C, ndiyeno pamene kutentha kumachepa, mtengo wa Ct umakhala waukulu.Mosiyana ndi zimenezi, pamene kutentha kumawonjezeka, dongosolo lachiwiri limatsegulidwa ndipo mphamvu yowonjezera imawonjezeka.Mukafika pa kutentha kwina, kuonjezera kutentha sikungawongolere kukulitsa bwino.Chifukwa zoyambira sizingaphatikizidwe mokhazikika panthawiyi.Chifukwa chake,yang'anani kutentha ndi mtengo wotsika kwambiri wa Ct, komwe ndi kutentha kwabwino kwambiri pakukulitsa template yachiwiri!Inde, opusa anzeru ayenera kudziwa kuti ngati sikofunikira, ndi bwino kusintha zoyambira ndikupewa gawo lachiwiri.
5. Ntchito mlingo
MIQE—Kusanthula Zambiri
Kusanthula kwa data kumaperekedwa makamaka ndi fulorosenti kuchuluka kwa PCR chida.M'nkhani yapitayi, ntchito zambiri zowunikira deta zachitika, monga kulamulira kopanda kanthu, komwe kwafotokozedwa pakupanga kuyesera.Majini ofotokozera mkati, manambala obwereza, ndi zina zotero zafotokozedwa., apa tikufotokozera makamaka kugwiritsa ntchito qPCR.
qPCR imagwiritsidwa ntchito kwambiri, ndipo kutsimikizira koyeserera ndi kuwunika kwa nucleic acid ndizomwe zimagwiritsidwa ntchito kwambiri.
mtheradi quantification
Logi (kukhazikika koyambirira) ili ndi ubale wofananira ndi kuchuluka kwa mizere.Njira yokhotakhota imatha kutengedwa kuchokera ku mulingo wokhala ndi nambala yoyambira yodziwika, ndiye kuti, kulumikizana kwa mzere wamachitidwe okulitsa kumatha kupezeka.Malingana ndi mtengo wa Ct wa chitsanzo, chiwerengero cha chitsanzocho chikhoza kuwerengedwa.Kuchuluka kwa ma templates kuti muphatikizepo.
Njira Yowerengera Yokwanira Kwambiri
Kuchulukitsidwa kotheratu kuyenera kukhazikitsidwa pamapindikira okhazikika.Kuti mupange kapindika wokhazikika, muyezo umafunika.Nthawi zambiri, muyezo ndi plasmid yomwe imapezeka popanga jini yomwe mukufuna.Chifukwa chiyani ndi plasmid?Chifukwa DNA ya plasmid yozungulira ndiyokhazikika kwambiri.Chepetsani chinthu chokhazikika mu 5 mpaka 6 gradient molingana ndi kuwirikiza kawiri (10-fold dilution), ndipo tcherani khutu ku kufananako pochepetsa.Lolani mtengo wa Ct ukhale pakati pa 15-30.
Kukonzekera kokhazikika
Panthawi imodzimodziyo, chitsanzo choyesedwa chiyeneranso kuchepetsedwa moyenerera (kumbukirani dilution factor), ndipo mtengo wa Ct uyeneranso kugwera pakati pa 15-30.Zogulitsa zokhazikika + zitsanzo zoyesedwa zimayikidwa pamakina pamodzi.Pambuyo pa kuthamanga, phokoso lokhazikika linapangidwa ndi chinthu chokhazikika, ndipo zitsanzo zomwe ziyenera kuyesedwa zinabweretsedwa muzitsulo zokhazikika kuti ziwerengetsedwe.
Hepatitis B virus HBV quantification ndi momwe mungawerengere kuchuluka kwa kachilomboka mu 1ml yamagazi.
Kuwerengera nambala ya kopi
Zitsanzo zoyesedwa kuti ziyesedwe (ng/ul) = OD260 × 50ug/ml × dilution factor
Zitsanzo za kulemera kwa maselo = chiwerengero cha maziko × 324
Nambala yachitsanzo yoyesedwa (makope / ul) = kuchuluka kwa zitsanzo kuti ziyesedwe / kulemera kwachitsanzo × 6 × 1014
Njira yowerengera nambala yakopi
Zomwe zili pamwambazi ndi njira yowerengera kuti mudziwe kuchuluka kwake.Ili ndi vuto la masamu lomwe lingathe kuthetsedwa mukamaliza sukulu ya sekondale, ndipo mavuto a masamu nthawi zambiri amathetsedwa ndi makompyuta.Ngati simukumvetsa, mutha kubwera kudzalankhulana.
quantification wachibale
Kuchuluka kwachibale kumagwiritsidwa ntchito makamaka mu kafukufuku wa sayansi.Ndi mavairasi angati omwe ali mu 1ml ya magazi, ndipo ndi kachilombo ka DNA, ichi ndi chochitika chodziwika bwino: kuchuluka kwa magazi kungadziwike, ndipo kachilombo ka DNA kamakhala kokhazikika.Komabe, zimakhala zovuta kwa ife kuyerekeza chiwerengero cha makope olembedwa a jini inayake patsamba, chifukwa n'zovuta kudziwa kukula, kulemera, ndi kukoma kwa tsamba, kuchuluka kwa RNA yotengedwa kumakhala kovuta kudziwa, komanso mphamvu ya kusindikiza kumbuyo kumakhala kovuta kudziwa, ndiko kunena kuti, sitepe iliyonse ingapangitse deta yoyesera kukhala ndi nsikidzi ndipo sangathe kugwiritsidwa ntchito.
Chifukwa chake, quantification yachibale iyenera kuyambitsa chinthu:jini yolozera mkati .
Mwa kuyankhula kwina, kuwerengera kwachibale kwenikweni ndikufanizira pakati pa jini yowunikira ndi jini yofotokozera mkati.Poyerekeza ndi minofu yomweyi ndi selo lomwelo, chikoka cha kukula kwachitsanzo, kuchuluka kwa RNA m'zigawo, zolembera zosinthika, komanso mphamvu ya PCR ndizochepa.Chifukwa cha kukula kwachitsanzo chaching'ono, chibadwa chamkati chamkati ndi majini omwe amawunikira adachepetsedwa.Ichi ndichifukwa chake takhala tikugogomezera kufanana ndi kukhazikika kale.
Ma genes a mkati amakhala ambirimajini osamalira nyumba(majini osunga m'nyumba), omwe amatanthawuza gulu la majini omwe amawonetsedwa mokhazikika m'maselo onse, ndipo zopangira zawo ndizofunikira kuti zisungidwe zoyambira zamoyo za ma cell.
Osasokoneza lingaliro ili.Majini osamalira m'nyumba ndi mawu ogwiritsiridwa ntchito kwachilengedwe, pomwe majini ofotokozera mkati ndi mawu oyesera.Majini osamalira m'nyumba amayenera kutsimikizira asanasankhidwe ngati majini ofotokozera mkati.
Mwachitsanzo, tidasankha majini angapo osamalira m'nyumba m'chithunzi chomwe chili pansipa kuti tiyese momwe amafotokozera m'maselo osiyanasiyana a minofu, ndipo tidapeza kuti milingo ya β-2-microglobulin inali yosiyana kwambiri ndi yamitundu ina itatu, kotero sakanatha kugwiritsidwa ntchito ngati majini ofotokozera mkati.
Pambuyo pomvetsetsa ntchito yowongolera ya jini yofotokozera zamkati, ma algorithms awiri amachokera chifukwa choyambitsa jini yolozera mkati.
·double standard curve njira
·2 – △△Njira ya Ct (njira yofananitsa mtengo wa CT)
Ngati mukufuna kuphunzira zamitundu ndi momwe majini amagwirira ntchito, chonde siyani kafukufuku wama algorithms ndikugwiritsa ntchito ma formula mwachindunji, kapena gwiritsani ntchito makina mwachindunji;ngati ndinu munthu wowongoka pa masamu ndi uinjiniya, chonde khalani omasuka.
double standard curve njira
Kuwerengera jini chandamale ndi jini yosungiramo nyumba yachitsanzo chowongolera ndi chitsanzo kuti chiyesedwe kudzera pamapindikira okhazikika, ndiyeno muwerengere mtengo wake molingana ndi chilinganizo chowerengera, chomwe ndi mulingo wofotokozera wachibale.
Ubwino: kusanthula kosavuta, kukhathamiritsa kosavuta koyesera
Zoyipa: Pa jini iliyonse, zoyeserera zilizonse ziyenera kupanga mayendedwe okhazikika
Kugwiritsa ntchito: Imodzi mwa njira ziwiri zomwe zimagwiritsidwa ntchito kwambiri komanso zodziwika bwino pakuwerengera malamulo a jini.
Fomula yake ndi iyi:
Zitsanzo ndi izi:
Werengetsani kuchuluka kwake potengera kuchuluka kwa zotsatira
2 – △△Ct njira (njira yofananitsa mtengo wa CT)
Ubwino wake: Palibe chifukwa chopanga njira yokhotakhota
Zoipa: Zimaganiziridwa kuti kukulitsa kwachangu kuli pafupi ndi 100%;kupatuka kokhazikika ndi <5%, ndipo mayendedwe okhazikika ndi magwiridwe antchito pakati pa kukulitsa kulikonse kumaganiziridwa kukhala kofanana;kukhathamiritsa kwa mikhalidwe yoyesera kumakhala kovuta kwambiri.
Kugwiritsa ntchito: Imodzi mwa njira ziwiri zomwe zimagwiritsidwa ntchito kwambiri komanso zodziwika bwino pakuwerengera malamulo a jini.
Zachidziwikire, kukulitsa bwino nthawi zambiri kumakhala kosatheka kukhala mwangwiro 1. Njira yowongolera: Ngati tikudziwa kuti jini yomwe mukufuna komanso jini yolozerayo ili ndi mphamvu yokulirapo yofanana, koma kukulitsa bwino sikuli kofanana ndi 1, ndiye kuti 2△△Ct ikhoza kuwongoleredwa monga: (1+E ) －△△Kulinganiza kwa Ct, mwachitsanzo, mwachitsanzo, Ct, 9, mwachitsanzo, ngati Ct. 1.95－△△Ct
Pakadali pano, zomwe zili pa fulorosenti kuchuluka kwa PCR zatha.
Nthawi yotumiza: Apr-06-2023
