Ⅰ. Wonjezerani mphamvu ya reaction system:

1. Siyanitsani RNA yapamwamba kwambiri:

Kuchita bwino kwa cDNA kumachokera ku RNA yapamwamba kwambiri.RNA yapamwamba kwambiri iyenera kuwonetsetsa kuti italikirapo ndipo ilibe zoletsa zomwe zilibe ma enzyme ojambulira, monga EDTA kapena SDS.Ubwino wa RNA umatsimikizira kuchuluka kwazinthu zomwe mungalembe ku cDNA.Njira yoyeretsera RNA ndi njira imodzi yogwiritsira ntchito isoocyanate/acidophenol.Pofuna kupewa kuipitsidwa kwa RNase, RNA yolekanitsidwa ndi chitsanzo cholemera mu RNase (monga kapamba) imafuna kusungidwa kwa formaldehyde kuti ipulumutse RNA yapamwamba kwambiri, yomwe imakhala yochuluka kwambiri posungira nthawi yaitali.RNA yotengedwa ku chiwindi cha makoswe idawonongeka pambuyo pa sabata imodzi yosungidwa m'madzi, pamene RNA yochokera ku makoswe inakhala yokhazikika pambuyo pa zaka zitatu zosungidwa m'madzi.Kuphatikiza apo, zolembedwa zazikulu kuposa 4kb ndizosavuta kutsata kuwonongeka kwa RNase kuposa zolemba zazing'ono.Pofuna kuonjezera kukhazikika kwa chitsanzo cha RNA chosungira, RNA ikhoza kusungunuka mu methalmamine ya ion, ndipo imasungidwa -70 ° C.Thylide yomwe imagwiritsidwa ntchito posunga RNA isakhale ndi zinthu zosiyanasiyana zomwe zimawononga RNA.RNA, yomwe imachokera ku kapamba, imatha kusungidwa mu methalmamine kwa chaka chimodzi.Mukakonzeka kugwiritsa ntchito RNA, mutha kugwiritsa ntchito njira zotsatirazi kuti muchepetse RNA: onjezani NaCl ku 0.2m ndi 4 kuchulukitsa kwa ethanol, kutentha kwa chipinda kwa mphindi 3-5, ndi 10,000 × g centrifugal kwa mphindi zisanu.

2. Gwiritsani ntchito reverse transcriptase popanda ntchito ya RNaseH (RNaseH-):

Ma RNase inhibitors nthawi zambiri amawonjezedwa kuti asinthe kalembedwe kuti awonjezere kutalika ndi zokolola za kaphatikizidwe ka cDNA.RNase inhibitor imawonjezedwa mumayendedwe oyamba a chain synthesis pamaso pa ma buffers ndi kuchepetsa othandizira monga DTT chifukwa pre-cDNA synthesis process denatures inhibitor, potero amamasula RNases omangidwa omwe amawononga RNA.Mapuloteni RNase inhibitor amangolepheretsa kuwonongeka kwa RNA ndi RNase A, B, C, ndipo samalepheretsa RNase pakhungu, choncho kusamala kuyenera kuchitidwa kuti musayambitse RNases kuchokera ku zala ngakhale kugwiritsa ntchito izi zoletsa.

Reverse transcriptase imathandizira kutembenuka kwa RNA kukhala cDNA.Onse M-MLV ndi AMV ali ndi endogenous RNaseH zochita kuwonjezera pa polymerase zochita zawo.Ntchito ya RNaseH imapikisana ndi ntchito ya polymerase ya zingwe za heterozygous zomwe zimapangidwa pakati pa ma tempulo a RNA ndi zoyambira za DNA kapena zingwe zowonjezera za cDNA, ndikuwononga RNA: zingwe za RNA mu ma complexes a DNA.Ma tempulo a RNA odetsedwa ndi ntchito ya RNaseH sangathenso kugwiritsidwa ntchito ngati magawo othandiza a cDNA synthesis, kuchepetsa zokolola ndi kutalika kwa kaphatikizidwe ka cDNA.Choncho kuchotsa kapena kuchepetsa kwambiri ntchito ya RNaseH ya reverse transcriptase kungakhale kopindulitsa kwambiri.

SuperScriptⅡ reverse transcriptase, MMLV reverse transcriptase ya RNaseH- ndi thermoScript reverse transcriptase, AMV ya RNaseH- inapereka cDNA yaitali kwambiri kuposa MMLV ndi AMV.Kukhudzidwa kwa RT-PCR kumakhudzidwa ndi kuchuluka kwa cDNA yopangidwa.ThermoScript ndizovuta kwambiri kuposa AMV.Kukula kwa zinthu za RT-PCR kumachepa chifukwa cha kuthekera kwa reverse transcriptase kupanga cDNA, makamaka popanga ma Cdna akulu.Poyerekeza ndi MMLV, SuperScripⅡ idakulitsa kwambiri zokolola zazinthu zazitali za RT-PCR.RNaseH-'s reverse transcriptase imawonjezeranso kukhazikika kwamafuta, kotero zomwe zimachitika zimatha kuchitika pa kutentha kwambiri kuposa 37-42 ℃.Pansi pa kaphatikizidwe kakulidwe, zoyambira za oligo(dT) ndi 10μCi [alpha-p]dCTP zidagwiritsidwa ntchito.Kupanga kwathunthu kwa unyolo woyamba kunawerengedwa pogwiritsa ntchito njira yamvula ya TCA.cDNA yautali wonse idawunikidwa pogwiritsa ntchito kuchotsa mizere yosankhidwa kukula ndikuwerengera mu gel ya alkaline agarose.

3. Wonjezerani kutentha kosungirako kutentha kwa zolembera mobwerera:

Kutentha kwakukulu kumathandizira kutsegula mawonekedwe achiwiri a RNA ndikuwonjezera zokolola za zomwe zimachitika.Kwa ma templates ambiri a RNA, kugwira RNA ndi primer pa 65 ° C popanda buffer kapena mchere ndiyeno kuziziziritsa mofulumira pa ayezi kumathetsa nyumba zambiri zachiwiri ndikulola kuti zoyambazo zimange.Komabe, ma templates ena akadali ndi mawonekedwe achiwiri, ngakhale pambuyo pakutentha kwamafuta.Kukulitsa ma tempuleti ovutawa kumatha kuchitidwa pogwiritsa ntchito ThermoScript reverse transcriptase ndikuyika reverse transcriptase reaction pa kutentha kwakukulu kuti mukweze bwino.Kutentha kwapamwamba kungathenso kuonjezera kutsimikizika, makamaka pamene cDNA synthesis ikuchitika pogwiritsa ntchito gene-specific primers (GSPS) (onani Mutu 3).Ngati mukugwiritsa ntchito GSP, onetsetsani kuti mtengo wa Tm woyambira ndi wofanana ndi kutentha komwe kumayembekezeredwa.Osagwiritsa ntchito oligo(dT) ndi zoyambira mwachisawawa pamwamba pa 60 ℃.Zoyambira zosasinthika ziyenera kuchitika pa 25 ℃ kwa mphindi 10 zisanachuluke mpaka 60 ℃.Kuphatikiza pakugwiritsa ntchito kutentha kwa reverse transcript, kukhazikika kumatha kusinthidwa ndikusamutsa mwachindunji kusakaniza kwa RNA / primer kuchokera pa 65 ℃ kutentha kwa denaturing kupita ku reverse transcript yosunga kutentha ndikuwonjezera preheated 2 × reaction mix (cDNA thermal initiation synthesis).Njirayi imathandiza kupewa kuphatikizika kwa ma intermolecular base komwe kumachitika pa kutentha kochepa.Kugwiritsa ntchito chida cha PCR kumathandizira kusintha kutentha komwe kumafunikira pa RT-PCR.

Tth kutentha kokhazikika kwa polymerase kumakhala ngati DNA polymerase pamaso pa Mg2+ ndi RNA polymerase pamaso pa Mn2+.Imatha kusunga kutentha mpaka 65 ℃.Komabe, kukhalapo kwa Mn2 + pa PCR kumachepetsa kukhulupirika, zomwe zimapangitsa Tth polymerase kukhala yosayenerera kukulitsa mwatsatanetsatane, monga cDNA cloning.Kuonjezera apo, Tth sichigwira ntchito bwino polemba m'mbuyo, zomwe zimachepetsa kukhudzidwa, ndipo popeza puloteni imodzi imatha kulembera mosinthana ndi PCR, machitidwe owongolera popanda reverse transcript sangathe kugwiritsidwa ntchito kusiyanitsa zinthu zokulitsa za cDNA ndi za DNA yoipitsidwa.

4. Zowonjezera zomwe zimalimbikitsa kumasulira mobwerera m'mbuyo:

Kuphatikiza kwa zowonjezera, kuphatikizapo glycerin ndi DMSO, ku njira yoyamba ya kaphatikizidwe kake kumatha kuchepetsa kukhazikika kwa nucleic acid double strand ndikumasula RNA yachiwiri.Mpaka 20% glycerin kapena 10% DMSO akhoza kuwonjezeredwa popanda kukhudza ntchito ya SuperScriptⅡ kapena MMLV.AMV imathanso kulekerera mpaka 20% glycerol popanda kuchepetsa ntchito.Kuti muwonjezere kukhudzika kwa RT-PCR mu SuperScriptⅡ reverse transcript reaction, 10% glycerol ikhoza kuwonjezeredwa ndikusungidwa pa 45 ℃.Ngati 1/10 ya mankhwala a retrotranscript-reaction awonjezeredwa ku PCR, kuchuluka kwa glycerol muzochita zokulitsa ndi 0.4%, zomwe sizokwanira kuletsa PCR.

5. RNaseH processing:

Kukhudzika kumatha kupitilizidwa pochiza kaphatikizidwe ka cDNA ndi RNaseH isanachitike PCR.Kwa ma templates ena, amaganiziridwa kuti RNA mu cDNA synthesis reaction imalepheretsa kumangiriza kwa zinthu zokulitsa, momwemo chithandizo cha RNaseH chingawonjezere chidwi.Nthawi zambiri, chithandizo cha RNaseH chimafunika pakukulitsa template ya chandamale ya cDNA yotalikirapo, monga tuberous scherosisⅡ yokhala ndi makope ochepa.Pa template yovutayi, RNaseH idakulitsa chizindikiro chopangidwa ndi cDNA chopangidwa ndi SuperScriptⅡ kapena AMV.Pazochita zambiri za RT-PCR, chithandizo cha RNaseH ndi chosankha chifukwa 95 ℃ insulated PCR denaturation sitepe nthawi zambiri imatulutsa RNA kuchokera ku RNA: DNA complex.

6. Njira zotsogola zodziwira zochepa za RNA:

RT-PCR imakhala yovuta makamaka ngati pali RNA yochepa chabe.Kuwonjezera kwa glycogen monga chonyamulira panthawi ya kupatukana kwa RNA kumathandiza kuonjezera zokolola zazing'ono.Glycogen yopanda RNase ikhoza kuwonjezeredwa nthawi yomweyo monga Trizol.Glycogen imasungunuka m'madzi ndipo imatha kukhalabe m'madzi ndi RNA kuti ithandizire kugwa kwamvula.Mlingo woyenera wa RNase-free glycogen ndi 250μg/ml kwa zitsanzo zosakwana 50mg za minofu kapena maselo 106 otukuka.

Kuphatikizika kwa acetylated BSA kuti musinthe machitidwe olembera pogwiritsa ntchito SuperScriptⅡ kumatha kukulitsa kukhudzika, komanso kwa RNA pang'ono, kuchepetsa kuchuluka kwa SuperScriptⅡ ndikuwonjezera mayunitsi 40 a RnaseOut nuclease inhibitor kumatha kuwongolera kuchuluka kwa kuzindikira.Ngati glycogen imagwiritsidwa ntchito pakulekanitsa kwa RNA, kuwonjezera kwa BSA kapena RNase inhibitors kuti musinthe machitidwe olembera pogwiritsa ntchito SuperScriptⅡ kumalimbikitsidwabe.

Ⅱ. Onjezani kutsimikizika kwa RT-PCR

1. cNDA kaphatikizidwe:

Njira zitatu zosiyana zingagwiritsidwe ntchito poyambitsa kaphatikizidwe ka chingwe cha cDNA choyamba, ndipo tsatanetsatane wa njira iliyonse zimakhudza kuchuluka ndi mtundu wa cDNA wopangidwa.

Njira yosasinthika ndiyo njira yocheperako mwa njira zitatuzi.Zoyambira zimasungidwa pamasamba angapo polembapo kuti apange cDNA yayifupi, yocheperako.Njirayi imagwiritsidwa ntchito nthawi zambiri kupeza ma 5′ terminal sequences ndi cDNA from RNA templates with secondary structural regions or with terminging sites that reverse transcriptase cannot replicate.Kuti mupeze cDNA yayitali kwambiri, chiŵerengero cha zoyambira ku RNA mu RNA iliyonse sampuli ziyenera kutsimikiziridwa mwamphamvu.Kuchuluka koyambirira kwa zoyambira mwachisawawa kumayambira 50 mpaka 250ng pa 20μl reaction system.Chifukwa cDNA yopangidwa kuchokera ku RNA yonse pogwiritsa ntchito zoyambira zosasinthika makamaka ndi ribosomal RNA, poly(A)+RNA nthawi zambiri imasankhidwa ngati template.

Kuyambika kwa Oligo (dT) ndikokhazikika kwambiri kuposa zoyambira mwachisawawa.Imasakanikirana ndi mchira wa poly(A) womwe umapezeka kumapeto kwa 3′ mRNA m'maselo ambiri a eukaryotic.Chifukwa poly(A)+RNA ndi pafupifupi 1% mpaka 2% ya RNA yonse, kuchuluka ndi zovuta za cDNA ndizochepa kwambiri kuposa ngati zida zoyambira zidagwiritsidwa ntchito.Chifukwa cha mawonekedwe ake apamwamba, oligo(dT) nthawi zambiri safuna kukhathamiritsa kwa RNA kupita ku primer ratio ndi poly(A)+ kusankha.Ndibwino kugwiritsa ntchito 0.5μg oligo(dT) pa 20μl reaction system.oligo(dT)12-18 ndi oyenera RT-PCR ambiri.ThermoScript RT-PCR System imapereka oligo(dT)20 chifukwa cha kukhazikika kwake kwamafuta ndipo ndiyoyenera kutentha kwambiri.

Gene-specific primers (GSP) ndi zoyambira zabwino kwambiri zosinthira mobwereza.GSP ndi oligonucleoside ya antisense yomwe imatha kusakanikirana ndi ma RNA komwe ikupita, m'malo mophatikiza ma Rna onse ngati zoyambira mwachisawawa kapena oligo(dT).Malamulo omwe amagwiritsidwa ntchito popanga zoyambira za PCR amagwiranso ntchito pamapangidwe a reverse transcription reaction GSP.GSP ikhoza kukhala yotsatizana ngati choyambira chokulitsa chomwe chimatsekeredwa kumapeto kwa mRNA3′, kapena GSP ikhoza kupangidwa kuti ipitirire kunsi kwa mtsinje ndi reverse amplification primer.Pazinthu zina zokulitsa, ndikofunikira kupanga zoyambira zotsutsana ndi chimodzi za RT-PCR yopambana chifukwa mawonekedwe achiwiri a chandamale cha RNA atha kuletsa choyambira kuti chisamange.Iwo amati kugwiritsa ntchito 1pmol antisense GSP mu unyolo kaphatikizidwe kaphatikizidwe dongosolo loyamba la 20μl.

2. Wonjezerani kutentha kosungirako kutentha kwa zolembera mobwerera:

Kuti mupindule mokwanira ndi kutsimikizika kwa GSP, reverse transcriptase yokhala ndi kukhazikika kwamafuta ambiri iyenera kugwiritsidwa ntchito.Heat-stable reverse transcriptase imatha kutsekedwa ndi kutentha kwambiri kuti iwonjezere kukhwima.Mwachitsanzo, ngati GSP ndi annealed pa 55 ° C, ndiye yeniyeni ya GSP si ntchito mokwanira ngati m'mbuyo transcript ikuchitika pa 37 ° C ndi otsika kwambiri kugwiritsa ntchito AMV kapena M-MLV.Komabe, SuperScripⅡ ndi ThermoScript imatha kuchitapo kanthu pa 50 ℃ kapena kupitilira apo, zomwe zimachotsa zinthu zosafunikira zomwe zimapangidwa potentha kwambiri.Kuti mudziwe zambiri, kusakaniza kwa RNA / primer kumatha kusamutsidwa mwachindunji kuchokera ku 65 ℃ kutentha kwa denaturation kupita ku reverse transcript yokhala ndi kutentha ndikuphatikiza ndi preheated 2 x reaction mix (kuyambitsa kutentha kwa cDNA synthesis).Izi zimathandiza kupewa kuyanjana kwapansi pakati pa mamolekyu pa kutentha kochepa.Kugwiritsa ntchito chida cha PCR kumathandizira kusintha kwa kutentha komwe kumafunikira pa RT-PCR.

3. Chepetsani kuipitsidwa kwa DNA:

Vuto limodzi lomwe lingakhalepo ndi RT-PCR ndikuti RNA imayipitsa DNA ya genomic.Kugwiritsa ntchito njira zabwino zolekanitsa za RNA, monga Trizol Reagent, kumachepetsa kuipitsidwa kwa DNA ya genomic mukukonzekera kwa RNA.Kupewa zinthu zopangidwa kuchokera ku genomic DNA, RNA imatha kuthandizidwa ndi amplification grade DnasⅠ kuchotsa DNA yoyipitsidwa isanalembedwenso.Zitsanzozo zidasungidwa pa 65 ℃ mu 2.0mM EDTA kwa mphindi 10 kuti athetse chimbudzi cha DNaseⅠ.EDTA chelates magnesium ions kuteteza magnesium ion yodalira RNA hydrolysis yomwe imapezeka pa kutentha kwambiri.

Kuti mulekanitse cDNA yokulirapo kuchokera kuzinthu zokulitsa za DNA, zoyambira zomwe zimalumikizana padera ndi exon yopatukana zitha kupangidwa.Zogulitsa za PCR zochokera ku cDNA zidzakhala zazifupi kuposa zomwe zimachokera ku DNA yowonongeka.Kuyesera kolamuliridwa kopanda kulembedwa m'mbuyo kumachitikanso pa template iliyonse ya RNA kuti muwone ngati chidutswa choperekedwa chikuchokera ku genomic DNA kapena cDNA.Zogulitsa za PCR zomwe zimapezedwa pakalibe zolembedwera zimachokera ku genome.

Zogwirizana nazo

RT-PCR Yosavuta^ᵀᴹIne (One Step)

-Chida cha gawo limodzi chimathandizira kuti zolembera zosinthika ndi PCR zizichitika mu chubu chomwecho.Zimangofunika kuwonjezera template ya RNA, zoyambira zenizeni za PCR ndi RNase-Free ddH₂O.

-Kusanthula kwanthawi yeniyeni kwa RNA kumatha kuchitika mwachangu komanso molondola.

-Zidazi zimagwiritsa ntchito makina apadera a Foregene reverse transcription reagent ndi Foregene HotStar Taq DNA Polymerase ophatikizidwa ndi njira yapadera yamachitidwe kuti apititse patsogolo kukulitsa bwino komanso momwe zimachitikira.

-The wokometsedwa kachitidwe kachitidwe zimapangitsa kuti zimene anachita kukhala apamwamba kuzindikira tilinazo, wamphamvu matenthedwe bata, ndi kulolerana bwino.

RT Easy II(Ndi GDNase) Master Premix For First-Strand CDNA Synthesis For Real Time PCR With GDNase

-Kutha kuchotsa gDNA, yomwe imatha kuchotsa gDNA mu template mkati mwa mphindi ziwiri.

-Njira yabwino yolembera, zimangotenga mphindi 15 kuti amalize kaphatikizidwe ka chingwe choyamba cha cDNA.

-Ma tempulo ovuta: ma tempulo okhala ndi GC yayikulu komanso mawonekedwe achiwiri ovuta amathanso kusinthidwa ndikuchita bwino kwambiri.

-High-sensitivity reverse transcript system, ma tempulo a pg-level amathanso kupeza cDNA yapamwamba kwambiri.

-Makina osindikizira a reverse ali ndi kukhazikika kwa kutentha kwambiri, kutentha kwabwino kwambiri ndi 42 ℃, ndipo akadali ndi ntchito yabwino yosinthira ku 50 ℃.