Kudziwika kwa kuzindikira

Nthawi zambiri, cholinga cha mapangidwe oyambira ndikukulitsa kutsimikizika kwa PCR.Izi zimatsimikiziridwa ndi mphamvu yochulukirapo kapena yocheperako yamitundu yambiri.Kusintha kumodzi kofunikira ndikutsatizana komwe kuli kumapeto kwa 3′ koyambira.

Chofunika kwambiri, zoyesa za PCR zopangidwira mwatsatanetsatane zimakhala ndi mwayi wogwiritsa ntchito bwino kwambiri pamitundu yosiyanasiyana, chifukwa kuyesako sikutulutsa zinthu zokulirapo mwapadera, potero kupikisana ndi PCR reagents kapena kuletsa kukulitsa kwakukulu.

Zoonadi, nthawi zina, kutsimikizika sikuli kofunikira kwambiri, mwachitsanzo, pamene cholinga ndikuwerengera okhudzana kwambiri koma tizilombo toyambitsa matenda, mapangidwe apadera, kukhathamiritsa ndi kutsimikizira miyezo imafunika.

Mphepete mwa kusungunula ndi njira yodziwika bwino yowunika momwe ma amplicon amadziwira, makamaka ponena za kukulitsa cholinga chimodzi.Komabe, ziyenera kutsindika kuti ma curve osungunuka amatha kusokeretsa chifukwa, mwachitsanzo, amatha kukhudzidwa ndi zotsatira zophatikizana za suboptimal primers ndi kutsika kwa template.

P5 |Mapiritsi osungunuka akuwonetsa kusintha kwa Tm komwe kumapezeka pazidziwitso ziwiri zamitundu iwiri ya DNA.

A. Pamalo okwera kwambiri (ad)), palibe chodziwikiratu choyezera pambuyo poyezera qPCR kumalizidwa.Pamene kusanja kwa template kumatsikira ku makope 50 (e), chinthu chomwe sichinali chachindunji chimayamba kuwoneka ndikukhala chinthu chokhacho chotsika kwambiri (f).

B. Mayesowa adalemba ma Tms omwewo pazolinga zonse, ndipo panalibe dimer yodziwikiratu ngakhale yotsika kwambiri (makopi asanu).Mukamagwiritsa ntchito njira ziwirizi zozindikirira, palibe zokulitsa zomwe zidapezeka mu NTCs.

P5 ikuwonetsa ma curve osungunula omwe amapezeka ndi zitsanzo momwe template imapezeka mosiyanasiyana.P 5a ikuwonetsa kuti pazigawo ziwiri zotsika kwambiri, ma Tms azinthu zokulitsa zomwe amapangidwa ndizochepa kuposa za amplicons enieni.

Mwachiwonekere, njira yodziwira iyi singagwiritsidwe ntchito modalirika kuti azindikire zolinga zomwe zimakhala zotsika kwambiri.

Chochititsa chidwi n'chakuti, ma NTC, mwachitsanzo, opanda DNA konse, sanalembe zinthu zokulitsa (zosatchulika), zomwe zikuwonetsa kuti maziko a genomic DNA amatha kutenga nawo gawo pakukulitsa/ku polymerization mosadziwika bwino.

Nthawi zina zoyambira zakumbuyo zotere komanso kukulitsa kosagwirizana kwenikweni sikungathe kukonzedwa, koma nthawi zambiri ndizotheka kupanga njira yodziwira yomwe ilibe kukweza kosagwirizana ndi template iliyonse ndi NTC (P 5b).

Apa, ngakhale kujambula kukulitsa kwazomwe mukufuna kutsata ndi Cq ya 35 kutulutsa njira yosinthira.Mofananamo, ma NTCs sanawonetse zizindikiro za kukulitsa kosagwirizana.Nthawi zina, machitidwe ozindikira amatha kudalira chakumwa cha mayiyo, ndipo kukulitsa kosagwirizana kwenikweni kumazindikirika muzolemba zina, zomwe zitha kukhala zokhudzana ndi kuchuluka kwa Mg2+.

Kukhazikika kwa kuzindikira

Kukhathamiritsa kwa Ta ndi gawo lothandiza pakutsimikizira kwamphamvu komanso kukhathamiritsa kwa kuzindikira kwa qPCR.Amapereka chisonyezero cholunjika cha kulimba kwa primer yokhazikitsidwa mwa kusonyeza kutentha (kapena kutentha) komwe kumapanga Cq yotsika kwambiri popanda kukulitsa NTC.

Kusiyanitsa kwawiri kapena kanayi pakukhudzidwa sikungakhale kofunikira kwa anthu omwe ali ndi mawu apamwamba a mRNA, koma poyesa kufufuza, kungatanthauze kusiyana pakati pa zotsatira zabwino ndi zabodza.

Makhalidwe a Ta a qPCR oyambira amatha kusiyanasiyana.Mayesero ena sakhala amphamvu kwambiri, ndipo ngati sakuchitidwa pansi pa mtengo woyenera wa Ta wa zoyambira, amagwa mwachangu.

Izi ndizofunikira chifukwa kudziwika kwamtunduwu nthawi zambiri kumakhala kovuta m'dziko lenileni, ndipo chiyero cha chitsanzo, kuchuluka kwa DNA, kapena kupezeka kwa DNA ina sikungakhale koyenera.

Kuonjezera apo, chiwerengero cha kopi chomwe mukufuna chikhoza kusiyana mosiyanasiyana, ndipo ma reagents, ziwiya zapulasitiki, kapena zida zingakhale zosiyana ndi zomwe zimagwiritsidwa ntchito poyambitsa mayesero.

P6|Kutentha kwa kutentha kumawonetsa kulimba kosiyana kwa kuzindikira kwa PCR.

A. Gwiritsani ntchito Bioline's Sensifast SYBR mastermix (kalozera nambala ya BIO-98050) kuti muyimbe PCR pa cDNA yokonzedwa kuchokera muubongo wamunthu wa RNA.

B. Gwiritsani ntchito chida cha CFX qPCR cha Bio-Rad kuti mujambule mapu okulitsa ndi kusungunuka kwa apalene (NM_033207, F: GCCATGGAGGAAAGTGACAGACC, R: CTCATGTGTGGGTGATCTCCTAGG).

C. Grafu yokulitsa ndi curve yosungunuka ya ACSBG1 (NM_015162.4, F: CTACACTTCCGGCACCACTGG, R: GTCCACGTGATATTGTCTTGACTCAG).

D. Girafu yokulitsa ndi kutha kwa GFAP (NM_002055.5, F: TGGAGAGGAAGATTGAGTCGCTGG, R: CGAACCTCCTCCTCGTGGATCTTC).

E. Cqs olembedwa pa kutentha kosiyanasiyana kwa annealing, kusonyeza kusiyana kwa Cq yolembedwa pansi pa kutentha kwa 7C.

P 6 ikuwonetsa zotsatira za mayeso osayenera, pomwe qPCR idachitidwa pogwiritsa ntchito gradient Tas pakati pa 59C ndi 67C (P 6a), pogwiritsa ntchito zoyambira zamitundu itatu yaubongo wamunthu.

Zitha kuwoneka kuchokera ku graph yokulitsa kuti Opalin primers ndiatali kwambiri chifukwa mtundu wawo wa Ta ndi wopapatiza kwambiri (Chithunzi 6b), ndiko kuti, ma Cqs amwazikana, zomwe zimapangitsa ma Cqs kufananizidwa kwambiri ndi ma Cqs awo abwino kwambiri.

Njira yodziwikirayi ndiyosakhazikika ndipo ingapangitse kukulitsa mopanda malire.Chifukwa chake, zoyambira ziwirizi ziyenera kukonzedwanso.Kuonjezera apo, kusanthula kwachitsulo chosungunula (kuyika) kumasonyeza kuti njira yeniyeni yodziwirayi ingakhalenso yovuta, chifukwa kusungunuka kwa Ta aliyense ndi kosiyana.

Njira yodziwira ACSBG1 yomwe ikuwonetsedwa mu P 6c ndi yolimba kwambiri kuposa njira yodziwira Opalin pamwambapa, koma ikadali kutali kwambiri, ndipo ndizotheka kuti ikhoza kuwongolera.

Komabe, tikugogomezera kuti palibe kugwirizana kofunikira pakati pa kulimba ndi kutsimikizika, chifukwa njira yowonongeka yomwe imapangidwa ndi njira yodziwikirayi imasonyeza mtengo womwewo wamtengo wapatali mu Tas zonse (inset).

Kumbali ina, kuyesa kwamphamvu kumakhala kolekerera kwambiri, kumapanga ma Cq ofanana mu Tas osiyanasiyana, monga muyeso wa GFAP wosonyezedwa mu P 6d.

Kusiyanitsa kwa Cqs komwe kumapezeka mumtundu womwewo wa 8 madigiri Celsius ndi osachepera 1, ndipo mphuno ya kusungunuka (inset) imatsimikizira zizindikiro zodziwika mu kutentha uku.Ndikoyenera kudziwa kuti Tas yowerengedwa ndi mtundu weniweni wa Ta ukhoza kukhala wosiyana kwambiri.

Pali malangizo ambiri opangidwa kuti athandize ochita kafukufuku kupanga zoyambira bwino, zambiri zomwe zimachokera ku malamulo omwe adakhazikitsidwa kale ndipo chidwi chambiri chaperekedwa ku 3′mapeto oyambira.Nthawi zambiri amalimbikitsidwa kuti aphatikizepo G kapena C kumapeto kwa 3' ndi maziko awiri a G kapena C (GC clamp), koma osaposa awiri mwa 5 otsiriza.

Mwachizoloŵezi, malamulowa akhoza kutsogolera ochita kafukufuku, koma sali olondola nthawi zonse.

P7 |Mapeto a 3'oyambira amakhala ndi zotsatira zochepa pazambiri kapena kuchita bwino.

A. Udindo wa zoyambira za jini ya HIF-1α (NM_181054.2).

B. Gwiritsani ntchito mowa wa Agilent Brilliant III SYBR Green mother mother (Cat. No. 600882) kukulitsa zinthu zisanu ndi chimodzi zoyesedwa.

C. Kukulitsa graph ndi curve yosungunuka yojambulidwa ndi chida cha CFX qPCR cha Bio-Rad ndi 3′end primers.Ma NTC amawonetsedwa mofiira.

D. Cqs zolemba zachiyeso chilichonse

Mwachitsanzo, zotsatira za P 7 zimatsutsana ndi lamulo la 3′end.Mapangidwe onse amatulutsa zotsatira zofanana, zophatikizira ziwiri zokha zomwe zimatsogolera kukulitsa kosagwirizana ndi NTC.

Komabe, sitingathe kuthandizira zotsatira za GC clip, chifukwa pamenepa, kugwiritsa ntchito A kapena T ngati maziko a 30 sikuchepetsa tsatanetsatane.

Yesani C, pomwe F primer imathera mu GGCC, idalemba ma Cqs mu NTCs, kuwonetsa kuti wina angafune kupewa izi kumapeto kwa 30.Tikugogomezera kuti njira yokhayo yodziwira mndandanda wabwino kwambiri wa 3′ wa gulu loyambira ndikuwunika moyeserera.

Kukulitsa luso

Chofunika kwambiri, ngakhale kudziwika kwa PCR kosadziwika kwenikweni sikungakhale kwachindunji, mphamvu yokulitsa imatha kusinthidwa ndikukulitsidwa m'njira zosiyanasiyana posintha ma enzyme, mowa wamayi, zowonjezera, ndi momwe amayendetsa njinga.

Kuti muwone momwe PCR imathandizira, ndi bwino kugwiritsa ntchito dilution ya 10 kapena 5 nthawi yomwe mukufuna nucleic acid, ndiko kuti, "njira yokhazikika".

Ngati ma amplicons a PCR kapena ma DNA opangira ma DNA agwiritsidwa ntchito popanga mapindikidwe wamba, ma serial dilutions a zolingazi ayenera kusakanizidwa ndi kuchuluka kwa DNA yakumbuyo (monga genomic DNA).

P8 |Dilution curve kuti muwone momwe PCR imathandizira.

A. Gwiritsani ntchito zoyambira za HIF-1: F: AAGAACTTTTAGGCCGCTCA ndi R: TGTCCTGTGGTGACTTGTCC ndi Agilent's Brilliant III SYBR Green mastermix (kalozera nambala 600882) ya PCR ndi mikhalidwe yosungunuka.

B. 100 ng RNA inasinthidwa, kuchepetsedwa ka 2, ndipo zitsanzo za cDNA zosungunuka zinachepetsedwa ka 5 ku 1 ng DNA yaumunthu.Njira yothirira ikuwonetsedwa pansipa.

C. Mawonekedwe a RT, dilution, ndi dilution serial adabwerezedwanso pa chitsanzo chachiwiri cha cDNA, ndipo zotsatira zake zinali zofanana.

P 8 imasonyeza mipiringidzo iwiri yokhazikika, pogwiritsa ntchito njira yodziwira yofanana pa zitsanzo ziwiri zosiyana za cDNA, zotsatira zake zimakhala zofanana, pafupifupi 100%, ndipo mtengo wa R2 umakhalanso wofanana, ndiko kuti, mlingo wa kuyenerera pakati pa deta yoyesera ndi mzere wobwereranso kapena deta Degree of linearity.

Mitundu iwiri yokhotakhota ndiyofanana, koma osati yofanana ndendende.Ngati cholinga ndikuwerengera molondola chandamale, ziyenera kudziwidwa kuti sikuloledwa kupereka nambala yowerengera popanda kufotokoza kusatsimikizika.

P9 |Kusatsimikizika koyezera komwe kumalumikizidwa ndi kuchulukana pogwiritsa ntchito curve yokhazikika.

A. Gwiritsani ntchito zoyambira za GAPDH (NM_002046) kuti muchite PCR ndi mapindikidwe osungunuka.F: ACAGTTGCCATGTAGACC ndi R: TAACTGGTTGAGCACAGG ndi Bioline's Sensifast SYBR mastermix (nambala ya catalog BIO-98050).

B. Tchati chokulitsa, piringidzo yosungunuka ndi mkhotolo wokhazikika wojambulidwa ndi chida cha Bio-Rad's CFX qPCR.

C. Standard curve graph ndi 95% confidence interval (CI).

D. Nambala ya kopi ndi 95% nthawi yodalirika ya ma Cq atatu omwe amachokera ku dilution curve.

P 9 ikuwonetsa kuti pakuyezetsa kokwanira, kusinthika kwachilengedwe kokhotakhota kumodzi kumakhala pafupifupi nthawi za 2 (95% nthawi yodalirika, yochepera mpaka pamlingo wapamwamba), womwe ukhoza kukhala kusiyana kwakung'ono komwe kungayembekezeredwe.

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