FastKing gDNA Kutaya RT SuperMix

A-1 RNA yawonongeka

—— Yeretsani RNA yapamwamba kwambiri popanda kuipitsidwa. Zinthu zomwe RNA imachotsedwa ziyenera kukhala zatsopano momwe zingatetezere kuwonongeka kwa RNA. Unikani umphumphu wa RNA pa gel osankhidwa musanayankhe RT. Pambuyo pochotsa RNA, iyenera kusungidwa mu 100% formamide. Ngati RNase inhibitor imagwiritsidwa ntchito, kutentha kumatentha kukhala <45 ° C, ndipo pH iyenera kukhala yochepera 8.0, apo ayi choletsa chimatulutsa RNase yonse yomangidwa. Kuphatikiza apo, RNase inhibitor iyenera kuwonjezeredwa mu mayankho omwe ali ndi ≥ 0.8 mM DTT.

A-2 RNA ili ndi zoletsa kusinthira kusindikiza

——Reverse transcription inhibitors akuphatikiza SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, guanidine salt, ndi zina zotero Sakanizani RNA yolamulira ndi nyerereyo, ndipo yerekezerani zokololazo ndi mphamvu ya RNA reaction kuti muwone ngati pali choletsa. Sambani mpweya wa RNA ndi 70% (v / v) ethanol kuti muchotse zoletsa.

A-3 Kusakwanira kwa zopangira zomwe zimagwiritsidwa ntchito popanga chingwe choyamba cha cDNA

—Dinani kuti kutentha kololeza ndi koyenera kwa zoyambira zomwe zagwiritsidwa ntchito poyesera. Kwa ma hexamers osasintha, tikulimbikitsidwa kuti tisunge kutentha pa 25 ° C kwa mphindi 10 musanafike kutentha. Pazomwe zimayambitsa ma jini (GSP), yesani ma GSP ena, kapena musinthe kupita ku oligo (dT) kapena hexamer yosasintha.

A-4 Kuchepa koyambira RNA

- Kuchulukitsa kuchuluka kwa RNA. Pazitsanzo za RNA zosakwana 50 ng, 0.1 μg mpaka 0.5 μg acetyl BSA itha kugwiritsidwa ntchito koyambirira kwa strand cDNA kaphatikizidwe

A-5 Zotsatirazi sizinafotokozeredwe m'matumba omwe anafufuzidwa.

——Yesani minofu ina.

A-6 PCR reaction imalephera

—Kwa RT-PCR ya magawo awiri, template ya cDNA yomwe ili mu PCR siyitha kupitilira 1/5 yama voliyumu.

A-1 Kukhazikitsa kosafunikira kwenikweni kwa ma primers ndi ma templates

—— Mapeto a 3'omaliza sayenera kukhala ndi 2-3 dG kapena dC. Gwiritsani ntchito zoyambira zapadera pa Gene muzitsulo zoyambirira m'malo mwa zoyambira kapena oligo (dT). Gwiritsani ntchito kutentha kochulukirapo koyambilira mzizunguliro zoyambirira, kenako kutentha kotsalira kotsalira. Gwiritsani ntchito kuyatsa kwa Taq DNA polymerase kwa PCR kuti musinthe momwe angachitire.

A-2 Mapangidwe oyipa amitundu yodziwika ndi majini

—Tsatirani mfundo zomwezi pakapangidwe kazinthu koyambira.

A-3 RNA yodetsedwa ndi genomic DNA

—Thandizani RNA ndi PCR-grade DNase I. Khazikitsani njira yoyeserera popanda kusinthanso kuti mupeze kuipitsidwa kwa DNA.

A-4 Kupanga choyambira

——Zipangidwe zoyambira popanda njira zowonjezera kumapeto kwa 3 '.

A-5 Mgulu wokwera kwambiri²⁺ ndende

—— Limbikitsani Mg²⁺ ndende ya template iliyonse ndi kuphatikiza koyambira

A-6 Yadetsedwa ndi DNA yakunja

——Gwiritsirani ntchito maupangiri osagwiritsa ntchito erosol ndi michere ya UDG.

A-1 Zomwe zili mu chingwe choyamba ndi zazikulu kwambiri

—— Chepetsani kuchuluka kwa chingwe choyambirira pamankhwala ochiritsira a PCR.

A-2 Kuchuluka kwambiri koyambira pamachitidwe a PCR

——Chepetsani zoyambira.

A-3 Zozungulira zambiri

—Opetsani mphamvu machitidwe a PCR ndikuchepetsa kuchuluka kwa kuzungulira kwa PCR.

A-4 Kutentha kotsika kwambiri

- Onjezani kutentha kokwanira kuti muchepetse kuyambitsa komanso kutambasula kosakhala kwapadera.

A-5 Kukulitsa kwapadera kwa zidutswa za oligonucleotide zopangidwa ndi DNase kuwonongeka kwa DNA -—Tengani RNA yapamwamba kwambiri yopewera kuipitsidwa kwa DNA.

RT-PCR ndiyokusinthira kusinthitsa RNA kukhala cDNA, kenako ndikugwiritsa ntchito cDNA yotsatidwayo ngati template ya PCR reaction kuti ikulitse chidutswacho. Sankhani zoyambira zosasinthika, Oligo dT ndi zoyambira zenizeni za jini kutengera mtundu wa kuyesaku. Zoyambira zonse zomwe zili pamwambazi zitha kugwiritsidwa ntchito ngati ma eukaryotic cell mRNA yopanda ma hairpin.

Zotsogola: Zoyenera RNA yayitali yokhala ndi zopangira tsitsi, komanso mitundu yonse ya RNA monga rRNA, mRNA, tRNA, ndi zina.

Oligo dT: Yoyenera RNA yokhala ndi PolyA tailing (prokaryotic RNA, eukaryotic Oligo dT rRNA ndipo tRNA ilibe michira ya PolyA). Chifukwa Oligo dT imalumikizidwa ndi mchira wa PolyA, mtundu wa zitsanzo za RNA umafunika kukhala wokwera, ndipo ngakhale kuwonongeka pang'ono kungachepetse kuchuluka kwa kapangidwe kakatundu ka cDNA.

Choyambirira chokhudza Gene: Chotsatira chotsatira cha template, choyenera pazochitika zomwe zikudziwika.

Pali njira ziwiri:

1. Njira yofotokozera mkati: Mwachidziwitso, cDNA ndi tizidutswa ta DNA totalikitsa mosiyanasiyana, chifukwa chake zotsatira za electrophoresis ndi smear. Ngati kuchuluka kwa RNA kuli kotsika, palibe chomwe chidzawonetsedwe mu electrophoresis, koma izi sizitanthauza kuti palibe chinthu chomwe chidzakwezedwa ndi PCR. Mwambiri, kutanthauzira kwamkati kungagwiritsidwe ntchito kuzindikira cDNA. Ngati zolembedwazo zili ndi zotsatira, mtundu wa cDNA ukhoza kutsimikizika (nthawi zingapo, ngati cholumikizira cha jini ndichotalikirapo, pangakhale zotsalira).

2. Ngati pali jini yodziwika yomwe ikukulitsidwa ndi template iyi, imatha kutsimikizika ndi oyambitsa a jiniyi. Kukulitsa kwamabuku amkati sikutanthauza kuti palibe vuto ndi cDNA. Chifukwa kutanthauzira kwamkati kuli ndi kuchuluka kwa cDNA, ndikosavuta kukulitsa. Ngati cDNA yawonongeka pang'ono pazifukwa zosiyanasiyana, kuchokera kuthekera, zotsatira za PCR zamajeremusi otsika kwambiri zimakhudzidwa kwambiri. Ngakhale kutanthauzira kwamkati kudakali kwakukulu, kukulitsa sikungakhudzidwe.

Kuwonongeka pang'ono kwa RNA. Onani kukhulupirika ndi kuyeretsa kwa RNA

Zomwe zili mu RNA zamitundu yosiyanasiyana zitha kukhala zosiyana, koma kwakukulu, RNA yonse yotulutsidwa iyenera kukhala ndimabande awiri omveka bwino a 28S ndi 18S mu gel electrophoresis, ndipo kuwala kwa gulu lakale kuyenera kupitilira kawiri kuposa kumeneku. Gulu la 5S likuwonetsa kuti RNA yawonongeka, ndipo kuwunika kwake ndikofanana ndi kuchuluka kwa kuwonongeka. Kukulitsa bwino kwa kutanthauzira kwamkati sikukutanthauza kuti palibe vuto ndi RNA, chifukwa zomwe zili mkati ndizochulukirapo, RNA imatha kukulitsidwa bola ngati kuwonongeka sikuli koopsa. OD₂₆₀/ OD₂₈₀Kuchuluka kwa RNA yoyera yoyezedwa ndi spectrophotometer kuyenera kukhala pakati pa 1.9 ndi 2.1. Kusayera pang'ono kwa mapuloteni mu RNA kumachepetsa kuchuluka. Malingana ngati mtengowo sutsika kwambiri, RT siyidzakhudzidwa. Chofunika kwambiri pa RT ndi umphumphu wa RNA.

Kukulitsa kwa jini yolozera mkati kumangowonetsa kuti RT yakwanitsa, koma sizogwirizana kwenikweni ndi mtundu wa chingwe cha cDNA. Chifukwa zidutswa zofotokozera zamkati nthawi zambiri zimakhala zazing'ono kukula komanso kufotokozera, ndizosavuta kuchita bwino pakusinthanso. Komabe, kukula ndi kufotokoza kwa chandamale cha jini kumasiyana pamitundu ina. Ubwino wa cDNA sungaweruzidwe kokha ndi kutengera kwamkati makamaka kwa zidutswa zomwe zikulimbana ndi 2 kb.

Zitsanzo zina zimakhala ndi nyumba zachiwiri zovuta, kapena zili ndi zolemera za GC, kapena ndizofunika ndizochepa. Nthawi izi, reverse transcriptase yoyenerera iyenera kusankhidwa kutengera kukula kwa chidutswacho ndi chitsanzocho. Kwa ma templates a RNA okhala ndi ma GC okwanira komanso mawonekedwe ovuta, zimakhala zovuta kutsegula mawonekedwe achiwiri potentha kwambiri, kapena ndi reverse transcriptase wamba. Kwa ma tempuletiwa, Quant Reverse Transcriptase itha kusankhidwa, chifukwa magwiridwe ake asintha bwino kuposa a M-MLV series reverse transcriptase, omwe amatha kusintha ma tempuleti osiyanasiyana a RNA moyenera ndikulemba RNA kukhala cDNA strand yoyamba pamlingo waukulu. Mukamagwiritsa ntchito chida chosinthira, 20 μl system itha kusintha kokha 1 μg yathunthu ya RNA. Chonde samalani ndi kuchuluka kwa zida za RT. Ngati templateyo iwonjezedwa mopitilira muyeso, kusindikiza kosinthika kumakomera RNA ndi kuchuluka kwambiri. Chifukwa chake, ndibwino kuti musapitirire kuchuluka kwadongosolo.

A-1 Dziwani ngati RNA yawonongeka kwambiri komanso ngati RT ikuyenda bwino

Mwambiri, chifukwa cholephera kukulitsa kwamkati nthawi zambiri kumayambitsidwa ndi kuwonongeka kwakukulu kwa RNA. Chifukwa china chotheka ndikusinthiratu kusindikiza. Zolemba zamkati sizingagwiritsidwe ntchito ngati muyezo kuweruza mtundu wa cDNA strand imodzi, koma itha kugwiritsidwa ntchito ngati muyezo wowunika ngati kusinthanso kwina kuli bwino ngati palibe vuto la mtundu wa RNA. Chofunika kwambiri pamasinthidwe ake ndikuteteza kutentha kosasinthasintha komanso kayendedwe kazomwe zimachitika kuti zithandizire kuyankha bwino.

A-2 Dziwani ngati zoyambira zokulitsira majini ofotokozera mkati ndizodalirika komanso ngati pali zovuta zilizonse ndi ma reagents omwe amagwiritsidwa ntchito mu PCR.

Pazowerengera zochepa, RNA iyenera kufotokozedweratu musanasinthe, zomwe zimafunikanso m'makina ambiri osindikizira, mwachitsanzo, kuyeza kuyika kwa RNA ngati 1 μg. Popeza cDNA yosindikizidwanso ndi njira yosakanikirana, kuphatikiza RNA, oligo dT, enzyme, dNTP, ngakhale zotsalira zochepa za DNA, kupatuka kudzayambika, chifukwa chake sikutheka kuwerengera cDNA molondola. Chifukwa chake, kuchuluka kwa RNA ndikofunikira. Poganizira momwe magwiridwe antchito amasinthira ndi ofanana pakati pa mitundu yosiyanasiyana, kuchuluka kwa cDNA komwe kumapezeka kuyenera kukhala kofananako, ndikuwunika kochulukirapo kumatha kuwonetsa kuyerekezera kwamanenedwe amitundu yosiyanasiyana pamlingo wofanana wa RNA. Pochita PCR yowerengera ya fluorescence, kuchuluka kwa cDNA sikungafunike mutasinthanso chifukwa mtundu wamkati umatha kutchulidwa.

Zimakhudzana kwambiri ndi majini, ndipo kusintha kwa chidutswa chachitali sikotheka m'mitundu yambiri. Choyamba, kuyeserera kosinthika ndikotsika kwambiri kuposa kwa PCR. Kachiwiri, dera lolemera la GC komanso mawonekedwe achiwiri amitundu yambiri amaletsa kusinthanso kosintha ndi PCR. Pomaliza, kudalirika ndi kukweza kwa PCR ndizovuta kutsimikizira nthawi yomweyo. Pakukonzanso, palibe amene angatsimikizire kuti atenga chidutswa chotalikirapo cha mitundu yotsika, makamaka pogwiritsa ntchito oligo dT. Ponena za 5 'UTR yokhala ndi GC yambiri, ndizovuta kwambiri. Chifukwa chake, ikadali njira yoyenera kusinthira zolembedwazo ndi zoyambira mwachisawawa, kupeza masamba achilengedwe omwe ali pachidutswacho, kukulitsa ndimagawo, kenako ndikuletsa chimbudzi ndi ligation. Mwambiri, kumakhala kovuta kukulitsa mwachindunji zidutswa zazikulu kuposa 2 kb, koma sizotheka nthawi zonse kupeza: 1. Choyamba, kutsimikizira kukhulupirika kwa RNA / mRNA, ndikuchotsa kwa TRIZOL. Chida cha 2.M-MLV RT-PCR chitha kugwiritsidwa ntchito mwachindunji. Onjezani nthawi yowonjezeretsa ndikuwonjezera nambala yazoyenda bwino pakukulitsa. Kapenanso, PCR yodzala ingagwiritsidwe, kapena kuchitapo kanthu kamodzi kapena kawiri koyamba ndi kutanthauzira koyenera komanso nthawi yowonjezera pasanafike kukweza kwa PCR, komwe kumathandizira kukulitsa zidutswa. Samalani kukhulupirika kwa polymerase. 3.Long Taq itha kugwiritsidwa ntchito mu PCR kuti ipeze zotsatira zabwino. 4. Pogwiritsa ntchito mapuloteni, kukhulupirika kwambiri kwa polymerase kuyenera kugwiritsidwa ntchito.

Pali mitundu iwiri ya reverse transcriptase yoperekedwa ndi TIANGEN: Quant / King RTase ndi TIANScript M-MLV. Kusiyanitsa kwakukulu pakati pawo ndikulowetsa ma templates. Quant ndi reverse transcriptase yapadera, yosiyana ndi M-MLV yomwe amagwiritsidwa ntchito kwambiri yochokera ku Moloney murine leukemia virus. Quant ndiwatsopano kwambiri yochita zinthu mobwerezabwereza yomwe imafotokozedwanso ndi uinjiniya Escherichia coli. Chiwerengero ndichabwino kukulitsa 50 ng-2 μg ya RNA yokhala ndi zochitika zotsika kwambiri komanso zokolola zambiri. Poyerekeza ndi MMLV wamba kapena AMV, mawonekedwe akulu kwambiri a Quant ndikuti ali ndi mgwirizano wamphamvu kwambiri ndi ma templates a RNA ndipo amatha kusinthira ma templates ovuta opanda kutentha kwakukulu. Kwa ma tempuleti okhala ndi ma GC apamwamba, magwiridwe antchito apamwamba ndi apamwamba. Komabe, reverse transcriptase ili ndi zochitika za RNase H, zomwe zingakhudze kutalika kwa cDNA product (yoyenera ma template a <4.5 kb). Potengera kusinthira kwachizolowezi, TIANScript MMLV reverse transcriptase ikulimbikitsidwa. RTase iyi ndi enzyme yosinthidwa yokhala ndi zochitika zochepa kwambiri za RNase H, zomwe ndizoyenera kutalikitsa (> 5 kb) cDNA kaphatikizidwe.

Gawo limodzi lokonzanso kusindikiza ndi kukweza kwa PCR kumamalizidwa mu chubu chimodzimodzi osatsegula chivundikirocho pakati pa cDNA kaphatikizidwe ndi kukulitsa, zomwe zimathandiza kuchepetsa kuipitsidwa. Popeza mitundu yonse ya cDNA yomwe idagwiritsidwa ntchito imagwiritsidwa ntchito kukulitsa, chidwi chake chimakhala chachikulu, osachepera 0.01 pg ya RNA yathunthu. Kuti RTPCR igwire bwino ntchito imodzi, mitundu yoyambira yokhudza jini imagwiritsidwa ntchito poyambitsa kaphatikizidwe ka cDNA. Njira ziwiri, zomwe ndi kusinthanso kosintha ndi kukweza kwa PCR zimachitika m'njira ziwiri. Choyamba kusindikiza kusanja kumachitika kuchokera mu template ya RNA kuti mupeze cDNA, ndipo cDNA yomwe imapezeka imayang'aniridwa ndi PCR imodzi kapena zingapo. Njira ziwirizi zitha kugwiritsa ntchito oligo (dT) kapena zoyambira mwachisawawa kuti zitsogolere kaphatikizidwe ka chingwe choyamba cha cDNA, ndipo chitha kusinthiratu kusindikiza chidziwitso chonse cha mRNA kuchokera pachitsanzo.