RT-qPCR imapangidwa kuchokera kuukadaulo wamba wa PCR.Imawonjezera mankhwala a fulorosenti (utoto wa fulorosenti kapena ma probe a fulorosenti) pamachitidwe achikhalidwe a PCR, ndikuzindikira njira yolumikizira PCR ndikuwonjezera munthawi yeniyeni molingana ndi makina awo osiyanasiyana owunikira.Kusintha kwa siginecha ya fluorescent pakatikati kumagwiritsidwa ntchito kuwerengera kuchuluka kwa kusintha kwazinthu pakusintha kulikonse kwa PCR.Pakadali pano, njira zodziwika bwino ndi njira ya utoto wa fulorosenti ndi njira yofufuzira.

Njira yopangira utoto wa fluorescent:
Utoto wina wa fulorosenti, monga SYBR Green Ⅰ, PicoGreen, BEBO, ndi zina zotero, sutulutsa kuwala paokha, koma umatulutsa fluorescence pambuyo pomangika ku kanjira kakang'ono ka dsDNA.Choncho, kumayambiriro kwa machitidwe a PCR, makinawo sangathe kuzindikira chizindikiro cha fulorosenti.Pamene zomwe zimapitirira ku annealing-extension (njira ziwiri) kapena siteji yowonjezera (njira zitatu), zingwe ziwiri zimatsegulidwa panthawiyi, ndi DNA polymerase yatsopano Panthawi ya kaphatikizidwe ka strand, ma molekyulu a fulorosenti amaphatikizidwa mu dsDNA yaing'ono poyambira ndi kutulutsa fulorosenti.Pamene kuchuluka kwa ma PCR akuchulukirachulukira, utoto wochulukirapo umaphatikizana ndi dsDNA, ndipo chizindikiro cha fulorosenti chimakulitsidwanso mosalekeza.Tengani SYBR Green Ⅰ monga chitsanzo.
Njira yofufuzira:
Taqman probe ndiye njira yomwe imagwiritsidwa ntchito kwambiri ndi hydrolysis.Pali gulu la fulorosenti kumapeto kwa kafukufuku wa 5′, nthawi zambiri FAM.Kufufuza komweko kumayenderana ndi jini yomwe mukufuna.Pali gulu lozimitsa fulorosenti kumapeto kwa 3'mapeto a fluorophore.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) Pamene chisangalalo sipekitiramu zikuchulukana ndi mtunda uli pafupi kwambiri (7-10000 molekyulu kuvomereza 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.

Chithunzi 1 Mfundo ya qRT-PCR

Kupanga koyambira
Mfundo Zazikulu:

Zoyambira ziyenera kupangidwa m'chigawo chosungidwa cha nucleic acid ndikukhala ndi tsatanetsatane.

Ndibwino kugwiritsa ntchito ndondomeko ya cDNA, ndipo ndondomeko ya mRNA ndiyovomerezeka.Ngati sichoncho, fufuzani ma CD a dera la DNA.
Kutalika kwa fulorosenti yochulukirachulukira ndi 80-150bp, yayitali kwambiri ndi 300bp, utali woyambira nthawi zambiri umakhala pakati pa 17-25 maziko, ndipo kusiyana pakati pa zoyambira zoyambira kumtunda ndi kumunsi sikuyenera kukhala kwakukulu.

Zolemba za G + C zili pakati pa 40% ndi 60%, ndipo 45-55% ndizo zabwino kwambiri.
Mtengo wa TM uli pakati pa madigiri 58-62.
Yesetsani kupewa ma dimers oyambira ndi ma self-dimers, (osawoneka kuposa ma 4 awiriawiri otsatizana otsatizana) kapangidwe ka hairpin, ngati kuli kotheka, pangani ΔG <4.5kJ/mol* Ngati simungathe kuonetsetsa kuti gDNA yachotsedwa panthawi yolemba reverse Clean, ndi bwino kupanga zoyambira za intron, C. kapangidwe kosalekeza (2-3) zoyambira ndi zosagwirizana
yeniyeni The homology of the heterogeneous amplified sequency makamaka osachepera 70% kapena ali ndi 8 complementary base homology.
Nawonsomba:
Kusaka kwa CottonFGD ndi mawu osakira
Kupanga koyambira:
IDT-qPCR primer design

Chithunzi cha Fig2 IDT pa intaneti chida choyambira chida

Mawonekedwe a masamba a Fig3
Mapangidwe a lncRNA oyambira:
lncRNA:masitepe omwewo monga mRNA.
miRNA:Mfundo ya njira ya tsinde-loop: Popeza ma miRNA onse ndi otsatizana afupikitsa pafupifupi 23 nt, kuzindikira kwachindunji kwa PCR sikungatheke, kotero chida chotsatizana ndi tsinde chimagwiritsidwa ntchito.Tsatanetsatane wa tsinde ndi DNA ya chingwe chimodzi cha pafupifupi 50 nt, yomwe imatha kupanga mapangidwe atsitsi okha.3 'Mapeto atha kupangidwa ngati kutsatizana kotsatizana ndi chidutswa cha miRNA, ndiye kuti chandamale cha miRNA chikhoza kulumikizidwa ndi mndandanda wa tsinde-loop panthawi yolemba mobwerera, ndipo kutalika kwake kumatha kufika 70bp, zomwe zimagwirizana ndi kutalika kwa chinthu chokulitsa chomwe chimatsimikiziridwa ndi qPCR.Kujambula koyambirira kwa miRNA.
Kuzindikira mwachindunji:
Pansi pa intaneti: CottonFGD kuphulika motsatizana
Kuphulika kwanuko: Fotokozerani kugwiritsa ntchito Blast + kuchita kuphulika kwanuko, linux ndi macos zitha kukhazikitsa mwachindunji nkhokwe yakomweko, Win10 system imathanso kuchitika mutakhazikitsa ubuntu bash.Pangani nkhokwe yakuphulika kwanuko ndi kuphulika kwanuko;tsegulani ubuntu bash pa win10.
Zindikirani: thonje la kumtunda ndi ku chilumba cha m'nyanja ndi mbewu za tetraploid, choncho zotsatira za kuphulika nthawi zambiri zimakhala machesi awiri kapena kuposerapo.M'mbuyomu, kugwiritsa ntchito ma CD a NAU ngati nkhokwe kuti mupangitse kuphulika ndikotheka kupeza majini awiri amtundu wamtundu wokhala ndi kusiyana kochepa kwa SNP.Nthawi zambiri, majini awiri a homologous sangathe kulekanitsidwa ndi mapangidwe oyambira, chifukwa chake amawonedwa ngati ofanana.Ngati pali indel yodziwikiratu, primer nthawi zambiri imapangidwira pa indel, koma izi zingayambitse dongosolo lachiwiri la primer Mphamvu yaulere imakhala yapamwamba, zomwe zimapangitsa kuchepa kwa mphamvu zowonjezera, koma izi sizingalephereke.

Kuzindikira koyambira kwachiwiri:
Masitepe:tsegulani oligo 7 → kutsatizana kwa template → kutseka zenera laling'ono → sungani → pezani choyambira pa template, dinani ctrl+D kuti muyike utali woyambira → santhulani zigawo zingapo zachiwiri, monga thupi lodziyimira pawokha, heterodimer, hairpin, mismatch, ndi zina zambiri.Chotsatira cha chiyambi cha kutsogolo ndi chabwino, palibe chodziwikiratu cha dimer ndi hairpin, palibe maziko owonjezera opitilira, ndipo mtengo wathunthu wa mphamvu yaulere ndi wocheperapo 4.5, pomwe choyambira chakumbuyo chikuwonetsa mosalekeza Maziko a 6 ndi othandizira, ndipo mphamvu yaulere ndi 8.8;kuonjezerapo, dimer yowonjezereka ikuwonekera kumapeto kwa 3, ndipo dimer ya 4 yotsatizana maziko ikuwonekera.Ngakhale mphamvu zaulere sizokwera, 3 ′ dimer Chl imatha kukhudza kwambiri katchulidwe kake komanso kukulitsa bwino.Komanso, m'pofunika kufufuza hairpins, heterodimers, ndi mismatches.

Zotsatira za Fig3 oligo7
Kuzindikira kokwanira kwa kukulitsa:
Kuchita bwino kwa machitidwe a PCR kumakhudza kwambiri zotsatira za PCR.Komanso mu qRT-PCR, kukulitsa bwino ndikofunikira kwambiri pazotsatira zochulukira.Chotsani zinthu zina, makina ndi ma protocol mu buffer reaction.Ubwino wa zoyambira umathandizanso kwambiri pakukulitsa bwino kwa qRT-PCR.Kuti muwonetsetse kuti zotsatira zake ndi zolondola, zonse zowerengera za fluorescence komanso kuwerengetsa kwathunthu kwa fluorescence ziyenera kuzindikira mphamvu yakukweza kwa zoyambira.Ndizodziwika kuti Kuchita bwino kwa qRT-PCR kukulitsa mphamvu kuli pakati pa 85% ndi 115%.Pali njira ziwiri:
1. Njira yopindika yokhazikika:
a.Sakanizani cDNA
b.Kuchuluka kwa gradient
c.qPCR
d.Linear regression equation kuti muwerengere kuchuluka kwa matalikidwe
2. LinRegPCR
LinRegPCR ndi pulogalamu yowunikira nthawi yeniyeni ya RT-PCR Data, yotchedwanso kuchuluka kwa PCR (qPCR) data yotengera SYBR Green kapena chemistry yofananira.Pulogalamuyi imagwiritsa ntchito deta yosasinthika yokhazikika, imapanga kuwongolera koyambira pachitsanzo chilichonse Payokha, imasankha zenera la mzere kenako amagwiritsa ntchito kusanthula kwa mzere kuti agwirizane ndi mzere wowongoka kudzera pa data ya PCR.Kuchokera pamtunda wa mzerewu mphamvu ya PCR ya chitsanzo chilichonse imawerengedwa.Kuthekera kwa PCR pa amplicon ndi mtengo wa Ct pachitsanzo chilichonse chimagwiritsidwa ntchito kuwerengera kuchuluka koyambira pachitsanzo chilichonse, chofotokozedwa m'mayunitsi a fluorescence.Kuyika kwa data ndi kutulutsa kumachitika kudzera mu Excel spreadsheet.Chitsanzo chokha
kusakaniza kumafunika, palibe gradient
njira zofunika:(Tengani Bole CFX96 mwachitsanzo, osati Machine yokhala ndi ABI yomveka)
kuyesa:ndi kuyesa kokhazikika kwa qPCR.
Kutulutsa kwa data ya qPCR:LinRegPCR imatha kuzindikira mitundu iwiri ya mafayilo otulutsa: RDML kapena quantification Amplification.M'malo mwake, ndiye nthawi yeniyeni yodziwikiratu nambala yozungulira ndi chizindikiro cha fluorescence ndi makina, ndipo kukulitsa kumapezedwa posanthula mtengo wakusintha kwa fulorosenti ya gawo lofananira bwino.
Kusankha deta: Mwachidziwitso, mtengo wa RDML uyenera kugwiritsidwa ntchito.Akuti vuto la kompyuta yanga ndikuti pulogalamuyo siyingazindikire RDML, chifukwa chake ndili ndi mtengo wotulutsa bwino kwambiri monga deta yoyambirira.Ndibwino kuti tichite kuwunika akhakula deta choyamba, monga kulephera kuwonjezera zitsanzo, etc. mfundo akhoza zichotsedwa mu deta linanena bungwe (kumene, inu simungakhoze kuchotsa iwo, LinRegPCR kunyalanyaza mfundo izi mu siteji yotsatira)

Fig5 qPCR kutumiza kunja kwa data

Fig6 kusankha zitsanzo za ofuna kusankha

Kuyika kwa data:Tsegulani zotsatira zokulitsa ziyeneretso.xls, → tsegulani LinRegPCR → fayilo → werengani kuchokera ku Excel → sankhani magawo monga momwe zasonyezedwera pa Chithunzi 7 → OK → dinani kuti mudziwe zoyambira

Fig7 masitepe a kulowetsa kwa data kwa linRegPCR

Zotsatira:Ngati palibe kubwereza, palibe gulu lomwe likufunika.Ngati pali kubwereza, gululo likhoza kusinthidwa mumagulu a zitsanzo, ndipo dzina la jini limalowetsedwa mu chizindikiritso, ndiyeno jini yomweyi idzagawidwa yokha.Pomaliza, dinani fayilo, tumizani excel, ndikuwona zotsatira.Kuchita bwino kwa kukulitsa ndi zotsatira za R2 pachitsime chilichonse zidzawonetsedwa.Kachiwiri, ngati mugawika m'magulu, kuwongolera bwino kwapakati kumawonekera.Onetsetsani kuti kukulitsa bwino kwa primer iliyonse kuli pakati pa 85% ndi 115%.Ngati ndi yayikulu kwambiri kapena yaying'ono kwambiri, zikutanthauza kuti kukulitsa bwino kwa primer ndikosavuta.

Chithunzi cha 8 Zotsatira ndi zotsatira za data

Njira yoyesera:
Zofunikira zamtundu wa RNA:
Chiyero:1.72.0 ikuwonetsa kuti pangakhale isothiocyanate yotsalira.Nucleic acid A260 / A230 yoyera iyenera kukhala yozungulira 2. Ngati pali kuyamwa kwamphamvu pa 230 nm, kumasonyeza kuti pali zinthu zakuthupi monga phenate ions.Kuphatikiza apo, imatha kuzindikirika ndi 1.5% agarose gel electrophoresis.Lozani chikhomo, chifukwa ssRNA ilibe denaturation ndipo molekyulu yolemetsa logarithm ilibe ubale wolumikizana, ndipo kulemera kwa maselo sikungafotokozedwe molondola.Kukhazikika: Mwamwanoayizosakwana 100ng/ul, ngati ndende ndi otsika kwambiri, chiyero zambiri otsika osati wamtali

Chithunzi cha 9 RNA gel

Kuonjezera apo, ngati chitsanzocho ndi chamtengo wapatali ndipo chiwerengero cha RNA ndi chachikulu, tikulimbikitsidwa kuti tiyitchule pambuyo pochotsa, ndikuchepetsera RNA kuti ikhale yomaliza ya 100-300ng / ul kuti isindikize.Munjira yolembera m'mbuyo, mRNA ikalembedwa, zoyambira za oligo (dt) zomwe zimatha kumangirira ku michira ya polyA zimagwiritsidwa ntchito polemba mobwerera, pomwe lncRNA ndi circRNA amagwiritsa ntchito zoyambira zosawerengeka za hexamer (Random 6 mer) polemba RNA yonse Kwa miRNA, zolemba zapadera za miRNA zimagwiritsidwa ntchito pakhosi.Makampani ambiri tsopano akhazikitsa zida zapadera za tailing.Kwa njira ya tsinde-loop, njira yopangira michira ndiyosavuta, yowonjezereka, komanso yopulumutsa reagent, koma Zotsatira za kusiyanitsa ma miRNA a banja lomwelo siziyenera kukhala zabwino monga njira ya tsinde-loop.Chida chilichonse cholembera m'mbuyo chimakhala ndi zofunikira pakuchulukira kwa ma jini enieni (malupu).Zomwe zimagwiritsidwa ntchito pa miRNA ndi U6.Pakutembenuka kwa tsinde-loop, chubu la U6 liyenera kusinthidwa padera, ndipo zoyambira zakutsogolo ndi zakumbuyo za U6 ziyenera kuwonjezeredwa mwachindunji.Onse a circRNA ndi lncRNA amatha kugwiritsa ntchito ma HKG ngati mafotokozedwe amkati.Mukuzindikira kwa cDNA,
ngati palibe vuto ndi RNA, cDNA iyeneranso kukhala yabwino.Komabe, ngati ungwiro wa kuyesera ukutsatiridwa, ndi bwino kugwiritsa ntchito jini yofotokozera mkati (Reference gene, RG) yomwe imatha kusiyanitsa gDNA ndi ma CD.Kawirikawiri, RG ndi jini yosamalira nyumba., HKG) monga momwe tawonetsera pa Chithunzi 10;Panthawiyo, ndinali kupanga mapuloteni osungira soya, ndikugwiritsa ntchito actin7 yomwe inali ndi introns ngati cholembera chamkati.Kukula kwa kachidutswa kokulirapo kwa pulayimale iyi mu gDNA kunali 452bp, ndipo ngati cDNA idagwiritsidwa ntchito ngati template, inali 142bp.Kenako zotsatira za mayeso zidapeza kuti Gawo la cDNA linali loipitsidwa ndi gDNA, ndipo zidatsimikiziranso kuti panalibe vuto ndi zotsatira za reverse transcript, ndipo zitha kugwiritsidwa ntchito ngati template ya PCR.Ndizopanda ntchito kuyendetsa agarose gel electrophoresis mwachindunji ndi cDNA, ndipo ndi gulu lofalikira, lomwe silikukhutiritsa.

Chithunzi cha 10 cDNA

Kutsimikiza kwa mikhalidwe ya qPCRnthawi zambiri palibe vuto malinga ndi ndondomeko ya zida, makamaka pa sitepe ya mtengo wa tm.Ngati zoyambira zina sizinapangidwe bwino panthawi yopanga mapangidwe, zomwe zimapangitsa kusiyana kwakukulu pakati pa mtengo wa tm ndi theoretical 60 ° C, tikulimbikitsidwa kuti cDNA Pambuyo pa zitsanzo zosakanikirana, yendetsani gradient PCR ndi zoyambira, ndipo yesetsani kupewa kuyika kutentha popanda magulu monga mtengo wa TM.

Kusanthula deta

The ochiritsira wachibale fluorescence kuchuluka PCR processing njira kwenikweni malinga 2-ΔΔCT.Data processing template.

Zogwirizana nazo:

Real Time PCR Easy^TM – Taqman

Real Time PCR Easy^TM -SYBR GREEN I

RT Easy I (Master Premix for first strand cDNA synthesis)

RT Easy II(Master Premix for first strand cDNA synthesis for qPCR)