I-FastKing gDNA isusa i-RT SuperMix

I-A-1 RNA ihlazekile

—Kucoca i-RNA ekumgangatho ophezulu ngaphandle kokungcola. Izinto ekukhutshelwa kuzo i-RNA kufuneka zibe zintsha kangangoko ukuthintela ukonakaliswa kwe-RNA. Hlaziya ukuthembeka kwe-RNA kwi-gel echongiweyo ngaphambi kokuphendula kwe-RT. Emva kokukhutshwa kweRNA, kufuneka igcinwe kwi-100% formamide. Ukuba kusetyenziswa i-RNase inhibitor, ubushushu bokufudumeza bube ngu- <45 ° C, kwaye i-pH kufuneka ibe ngaphantsi kwe-8.0, kungenjalo i-inhibitor iya kuyikhupha yonke i-RNase ebotshiweyo. Ngaphaya koko, i-RNase inhibitor kufuneka yongezwe kwizisombululo eziqukethe ≥ 0.8 mM DTT.

I-2-RNA iqulethe inhibitors zempendulo ekhutshelweyo ebhaliweyo

—— Ukuthintela ukukhutshelwa kwezithintelo kubandakanya i-SDS, i-EDTA, i-glycerol, i-sodium pyrophosphate, i-spermidine, i-formamide, i-guanidine ityuwa, njl. Hlamba imvula ye-RNA nge-70% (v / v) ethanol ukususa inhibitors.

A-3 Ukungonelisi okwaneleyo kwee-primers ezisetyenziselwa ukwenza umtya wokuqala we-cDNA

Misela ukuba iqondo lobushushu elongezelekileyo lilungele i-primers ezisetyenzisiweyo kulingo. Kwii-hexamers ezingahleliwe, kuyacetyiswa ukuba kugcinwe ubushushu kwi-25 ° C kangange-10 min ngaphambi kokufikelela kubushushu bokusabela. Kwizinto ezithile ezizodwa zemfuza (GSP), zama enye i-GSP, okanye tshintshela kwi-oligo (dT) okanye kwi-hexamer engahleliwe.

A-4 Inani elincinci lokuqalisa i-RNA

-Yandisa inani le-RNA. Kwiisampulu ze-RNA ezingaphantsi kwama-50 ng, i-0.1 μg ukuya kwi-0.5 μg i-acetyl BSA inokusetyenziswa kwi-strand yokuqala ye-cDNA synthesis

A-5 Ulandelelwano ekujoliswe kulo aluchazwanga kwizicubu ezihlalutyiweyo.

——Zama ezinye izicwili.

Ukuphendula nge-A-6 PCR kuyasilela

-Kwi-RT-PCR yamanyathelo amabini, itemplate ye-cDNA kwinqanaba lePCR ayinakodlula i-1/5 yevolumu yokuphendula.

A-1 Ukuncitshiswa okungachazwanga kwezinto zokuqala kunye neetemplate

-Isiphelo se-3'sokuqala asinakuba ne-2-3 dG okanye i-dC. Sebenzisa ii-primers ezikhethekileyo ze-Gene kwi-strand synthesis yokuqala endaweni ye-primers engahleliwe okanye i-oligo (dT). Sebenzisa amaqondo obushushu aphezulu annealing kwimijikelezo embalwa yokuqala, kwaye emva koko ubushushu obusongelayo obusezantsi. Sebenzisa i-Taq DNA polymerase eshushu eshushu ye-PCR ukuphucula ukubonakala kwempendulo.

A-2 Uyilo olubi lwama-primers athile akhethekileyo

Landela imigaqo efanayo yokuyilwa koyilo lokuqala.

I-A-3 RNA ingcoliswe yi-genomic DNA

Yiphathe i-RNA nge-PCR-grade DNase I. Cwangcisa indlela yokuphendula ngaphandle kokukhuphela umva ukufumanisa ukungcola kwe-DNA.

A-4 Ukuyilwa kwe-primer dimer

Uyilo lungavelanga ngokulandelelana kwisiphelo sesi-3.

A-5 Uphakamileyo kakhulu Mg²⁺ Uxinzelelo

—Yandisa uMg²⁺ Uxinzelelo lwetemplate nganye kunye nokudityaniswa kweprimer

A-6 Ungcoliswe yiDNA yangaphandle

—Sebenzisa iingcebiso ze-aerosol kunye nee-enzyme ze-UDG.

Umxholo wemveliso yokuqala yomtya uphezulu kakhulu

—Ukunciphisa inani lemveliso yokuqala yomtya kwinyathelo eliqhelekileyo lokuphendula kwePCR.

I-2-Inani eliphakamileyo kakhulu kwimpendulo ye-PCR

- - Nciphisa igalelo lokuqala.

A-3 Imijikelo emininzi kakhulu

Yandisa iimeko zokuphendula ze-PCR kunye nokunciphisa inani lomjikelo we-PCR.

A-4 Iqondo lobushushu elingaphantsi kakhulu

—Ukwandisa ubushushu bokongeza ukuthintela ulwaluko olungachazwanga kunye nolwandiso.

A-5 Ukwandiswa okungachazwanga kwamaqhekeza e-oligonucleotide aveliswe kukuthotywa kwe-DNase kwe-DNA -—Khupha i-RNA ekumgangatho ophezulu ukuthintela ungcoliseko lwe-DNA.

I-RT-PCR kukubuyisela umva ukukhuphela i-RNA kwi-cDNA, kwaye emva koko usebenzise i-cDNA ekhutshelweyo ebhalisiweyo njengetemplate yokuphendula kwe-PCR ukukhulisa isiqwenga ekujoliswe kuso. Khetha ii-primers ezingahleliwe, i-Oligo dT kunye ne-primers ezithile zemfuza ngokweemeko ezithile zovavanyo. Zonke izinto zokuqala zingasetyenziselwa iseli emfutshane ye-eukaryotic mRNA ngaphandle kwesakhiwo se-hairpin.

I-Random primer: Ilungele i-RNA ende enesakhiwo se-hairpin, kunye nazo zonke iintlobo ze-RNA ezinje nge-rRNA, mRNA, tRNA, njl.njl.

I-Oligo dT: Ilungele i-RNA kunye ne-PolyA tailing (i-prokaryotic RNA, i-eukaryotic i-Oligo dT rRNA kunye ne-tRNA ayinayo imisila ye-PolyA). Ngenxa yokuba i-Oligo dT ibotshelelwe kumsila wePolyA, umgangatho weesampulu ze-RNA kufuneka uphakame, kwaye nokuba lixabiso elincinci lokuthotywa liya kulinciphisa kakhulu inani lobungakanani be-cDNA synthesis.

I-primer ekhethekileyo ye-Gene: Iyahambelana nokulandelelana kwetemplate, efanelekileyo kwiimeko apho ulandelelwano ekujoliswe kulo luyaziwa.

Zimbini iindlela:

1. Indlela yereferensi yangaphakathi: Kwithiyori, i-cDNA ngamaqhekeza e-DNA obude obahlukeneyo, ke iziphumo ze-electrophoresis yi-smear. Ukuba ubuninzi be-RNA buphantsi, akukho mveliso iya kubonisa kwi-electrophoresis, kodwa oku akuthethi ukuba akukho mveliso iya kwandiswa yi-PCR. Ngokubanzi, ireferensi yangaphakathi inokusetyenziselwa ukufumana i-cDNA. Ukuba ireferensi yangaphakathi ineziphumo, umgangatho we-cDNA unokuqinisekiswa ngokusisiseko (kwiimeko ezimbalwa, ukuba isiqwenga semfuza ekujoliswe kuyo side kakhulu, kunokubakho ngaphandle).

2. Ukuba kukho imfuza eyaziwayo eyandisiweyo yile template, inokuqinisekiswa ngabokuqala bolu hlobo. Ukwandiswa kwesalathiso sangaphakathi akuthethi ukuba akukho ngxaki nge-cDNA. Kuba ireferensi yangaphakathi inentabalala ye-cDNA, kulula ukuyandisa. Ukuba i-cDNA ithobeke ngokuyinxenye ngenxa yezizathu ezahlukeneyo, ngokwembono yokunokwenzeka, iziphumo ze-PCR zezinto zofuzo ekujoliswe kuzo ziya kuchaphazeleka kakhulu. Ngelixa ireferensi yangaphakathi isephezulu ngobuninzi, ukukhula kwayo akunakuchaphazeleka.

Ukuthotywa kwenxalenye yeRNA. Fumana ingqibelelo kwaye ucoce i-RNA

Imixholo ye-RNA yeentlobo ezahlukeneyo inokwahluka, kodwa ngokubanzi, i-RNA iyonke ekhutshiweyo kufuneka iqule iibhendi ezimbini ezicacileyo ze-28S kunye ne-18S kwi-gel electrophoresis, kunye nokuqaqamba kwebhendi yangaphambili kufanele ukuba iphindwe kabini kunale yokugqibela. Ibhendi ye-5S ibonisa ukuba i-RNA ihlazisiwe, kwaye ukuqaqamba kwayo kuyalingana kwinqanaba lokuthotywa. Ukwandiswa ngempumelelo kwesalathiso sangaphakathi akuthethi ukuba akukho ngxaki kwi-RNA, kuba ireferensi yangaphakathi ikwindawo eninzi kakhulu, i-RNA inokuphakanyiswa ixesha elide de ukuthotywa kungabi nzima. I-OD₂₆₀/ OD₂₈₀Umyinge we-RNA emsulwa elinganiswa nge-spectrophotometer kufuneka ibe phakathi kwe-1.9 kunye ne-2.1. Inani elincinci lokungcola kweeprotheyini kwi-RNA liya kunciphisa umlinganiso. Logama ixabiso lingaphantsi kakhulu, i-RT ayizukuchaphazeleka. Eyona nto ibaluleke kakhulu kwi-RT yingqibelelo yeRNA.

Ukongezwa kohlobo lwesalathiso sangaphakathi kunokubonisa kuphela ukuba i-RT iphumelele, kodwa ayisiyiyo kwaphela enxulumene nomgangatho we-cDNA strand. Kungenxa yokuba iziqwenga zereferensi yangaphakathi zihlala zincinci ngobukhulu kwaye ziphezulu kwintetho, kulula ukuba ziphumelele ekubhaleni umva. Nangona kunjalo, ubungakanani kunye nokubonakaliswa kohlobo ekujoliswe kulo kwahluka ukusuka kuhlobo luye kuhlobo. Umgangatho we-cDNA awunakugwetywa kuphela ngokubhekiswa ngaphakathi ngokukodwa kumaqhekeza ekujoliswe kuwo angaphezulu kwe-2 kb.

Ezinye iisampulu zinezakhiwo ezimbaxa zasesekondari, okanye zinomxholo we-GC osisityebi, okanye zixabisekile ngentabalala ephantsi. Kule meko, i-reverse transcriptase efanelekileyo kufuneka ikhethwe ngokobungakanani beqhekeza ekujoliswe kulo kunye nesampulu. Kwiitemplate zeRNA ezinomxholo ophezulu we-GC kunye nolwakhiwo lwesekondari oluntsonkothileyo, kunzima ukuvula ulwakhiwo lwesibini kubushushu obuphantsi, okanye nge-reverse transcriptase eqhelekileyo. Kwezi tempilethi, iQuant Reverse Transcriptase inokuchongwa, kuba ukusebenza kwayo kokubhalwa umva ngokucacileyo kubhetele kunaleyo ye-M-MLV series reverse transcriptase, enokuthi ibuyisele ukuhanjiswa kweetemplate zeRNA ezahlukeneyo ngokufanelekileyo kwaye ikhuphele iRNA kwi-cDNA strand yokuqala ukuya kuthi ga kwinqanaba eliphezulu. Xa usebenzisa ikhithi yokubuyisela umva umva ngokubanzi, inkqubo ye-20 μl inokuguqula ngokupheleleyo ukubhalwa kwe-1 μg ye-RNA iyonke. Nceda unike ingqalelo ubuninzi be-RT yeseti. Ukuba itemplate yongezwa ngokugqithileyo, ukukhutshelwa okubhaliweyo kuya kuthanda i-RNA ngobuninzi obuphezulu. Ke ngoko, kungcono ukuba ungagqithi kubungakanani benkqubo.

A-1 Chonga ukuba ngaba i-RNA ihlazekile kakhulu kwaye ukuba i-RT iphumelele

Ngokubanzi, isizathu sokungaphumeleli kokunyuswa kwesalathiso sangaphakathi kuhlala kubangelwa kukuthotywa kweRNA. Esinye isizathu esinokubakho kukungaphumeleli kokukhutshelwa kokubhaliweyo. Isalathiso sangaphakathi asinakusetyenziswa njengomgangatho wokugweba umgangatho we-cDNA single strand, kodwa inokusetyenziswa njengomgangatho wokugweba ukuba ukukhutshelwa ngaphandle kuyaphumelela ukuba akukho ngxaki kumgangatho weRNA. Eyona nto ibaluleke kakhulu kwinkqubo yokubuyela umva kukugcina ubushushu obuqhubekayo kunye nenkqubo yokuphendula rhoqo ukuze kuphuculwe ukusebenza ngokukuko.

I-A-2 Chonga ukuba ngaba ii-primers zokuphucula uhlobo lwangaphakathi lwereferensi zithembekile kwaye ukuba kukho naziphi na iingxaki ngezixhobo ezisetyenziswa kwi-PCR.

Kwi-quantification ehambelana nayo, i-RNA kufuneka ibalwe ngaphambi kokubhalwa kwakhona, okufuneka kwakhona kwiikiti ezininzi zokukhuphela, umzekelo, ukulinganisa igalelo le-RNA njenge-1 μg. Kuba i-cDNA ekhutshelweyo ebuyiselweyo sisisombululo esixubeneyo, kubandakanya i-RNA, i-oligo dT, i-enzyme, i-dNTP, kunye nentsalela encinci ye-DNA, ukuphambuka kuya kubangelwa, ngenxa yoko akunakulinganiswa ngokuchanekileyo i-cDNA. Ke ngoko, ubungakanani beRNA buyimfuneko. Ukujonga ukusebenza ngokuchanekileyo kokukhutshelwa kuyafana phakathi kweesampulu ezahlukeneyo, isixa se-cDNA esifunyenweyo kufuneka silingane, kwaye uhlalutyo lobungakanani lunokubonisa ukuthelekiswa kwamanqanaba okubonisa ohlobo ahlukeneyo kwinani elifanayo leRNA iyonke. Xa usenza i-PCR ye-fluorescence ehambelanayo, i-cDNA yexabiso elinokungafuneki emva kokubhalwa kwakhona ngenxa yokuba uhlobo lwangaphakathi lwesalathiso lunokwenziwa njengesalathiso.

Inxulumene ikakhulu nemfuza, kwaye ukubuyela umva kwesiqwengana eside akunakwenzeka kwinkoliso yemfuza. Okokuqala, ukusebenza ngokuchanekileyo kokukhutshelwa kumazantsi kakhulu kunalawo e-PCR. Okwesibini, ummandla otyebileyo we-GC kunye nolwakhiwo lwesibini lweentlobo ezininzi zofuzo kuthintela kokubhalwa umva kokubuyela umva kunye nePCR. Okokugqibela, ukunyaniseka kunye nokunyusa ukusebenza kwePCR kunzima ukukuqinisekisa ngaxeshanye. Kwinkqubo yokukhuphela umva, akukho mntu unokuqinisekisa ukufumana iziqwengana ezinde zofuzo lwekopi esezantsi, ngakumbi usebenzisa i-oligo dT. Ngokuphathelele kwi-5 'ye-UTR ene-GC engaphezulu, kunzima ngakumbi. Ke ngoko, iseyindlela efanelekileyo yokubuyisela umva kokubhaliweyo kunye nezinto zokuqala ezingaqhelekanga, fumana iisayithi zokucoca indalo kwisiqwengana ekujoliswe kuso, wandise ngokwamacandelo, emva koko wenze isithintelo sokugaya kunye nokudibanisa. Ngokubanzi, kunzima ukukhulisa ngokuthe ngqo amaqhekeza amakhulu kune-2 kb, kodwa akusoloko kunzima ukufumana: 1. Okokuqala, qinisekisa ukuthembeka kwe-RNA / mRNA, kunye nokukhethwa kwe-TRIZOL. I-2.M-MLV RT-PCR kit ingasetyenziswa ngokuthe ngqo. Yandisa ixesha lokubamba kunye nokwandisa inani lomjikelo kwinkqubo yokwandisa ngokufanelekileyo. Ngenye indlela, i-PCR ehleliweyo inokusetyenziswa, okanye yenze isenzo esinye okanye ezibini kuqala ngokuchazwa ngokufanelekileyo kunye nexesha elandisiweyo ngaphambi kokukhulisa okuqhelekileyo kwePCR, enokunceda ukwandisa iziqwenga. Nika ingqalelo ukuthembeka kwepolymerase. 3.Long Taq inokusetyenziswa kwi-PCR ukufumana iziphumo ezifanelekileyo. Ukusetyenziswa kwesicelo sokubonisa iiprotein, kufuneka kusetyenziswe ukuthembeka okuphezulu kwepolymerase.

Zimbini iintlobo ze-reverse transcriptase ezinikezelwa yi-TIANGEN: I-Quant / King RTase kunye ne-TIANScript M-MLV. Umahluko ophambili phakathi kwabo linani lokufaka leetemplate. I-Quant yi-reverse transcriptase eyahlukileyo, eyahlukileyo kwi-M-MLV eqhelekileyo esetyenziswa kwi-Moloney murine virus ye-leukemia. I-Quant yinto ephezulu yokusebenza kakuhle kwe-transcriptase echazwe ngokutsha ngobunjineli i-Escherichia coli. Inani lilungele ukukhulisa i-50 ng-2 μg ye-RNA ngomsebenzi okhutshelweyo wokubuyela umva kunye nesivuno esikhulu. Xa kuthelekiswa neMMLV okanye i-AMV yesiqhelo, olona phawu lubalulekileyo kukuba inobuhlobo obomeleleyo neetemplate zeRNA kwaye inokubuyisa umva itemplate ezintsonkothileyo ngaphandle kobushushu obuphezulu. Kwiitemplate ezinomxholo ophezulu we-GC, ukusebenza ngokuchaseneyo kuphezulu. Nangona kunjalo, le transcriptase iphindayo inemisebenzi ye-RNase H, enokuthi ichaphazele ubude bemveliso ye-cDNA (efanelekileyo kwiitemplate ze <4.5 kb). Ukukhutshelwa okuqhelekileyo okubuyela umva kuyacetyiswa TIANScript MMLV reverse transcriptase. Le RTase yi-enzyme eguqulweyo enezinto ezibuthathaka kakhulu kwi-RNase H, efanelekileyo ixesha elide (> 5 kb) cDNA synthesis.

Inyathelo elinye lokukhutshelwa kokubuyela emva kunye nokukhulisa i-PCR kugqityiwe kwityhubhu enye ngaphandle kokuvula isiciko setyhubhu phakathi kokudityaniswa kwe-cDNA kunye nokukhulisa, oku kuluncedo ukunciphisa ukungcola. Kuba zonke iisampulu ze-cDNA ezifunyenweyo zisetyenziselwa ukukhulisa, ubuntununtunu buphezulu, ubuncinci be-0.01 pg ye-RNA iyonke. Ukuphumelela inyathelo elinye le-RTPCR, i-primers ezikhethekileyo zohlobo oluthile zisetyenziselwa ukuqala ukuhlanganiswa kwe-cDNA. Indlela enamanyathelo amabini, oko kukuthi ukubuyisela umva kokubhaliweyo kunye nokwandiswa kwePCR kwenziwa ngamanyathelo amabini. Okokuqala ukukhuphela ukuguqulela kwenziwa kwitemplate ye-RNA ukufumana i-cDNA, kwaye i-cDNA efunyenweyo ixhomekeke kwi-PCR enye okanye nangaphezulu. Indlela enamanyathelo amabini inokusebenzisa i-oligo (dT) okanye ii-primers ezingahleliwe ukukhokela ukudityaniswa komtya wokuqala we-cDNA, kwaye inokuguqula konke ukukhuphela ulwazi lwe-mRNA kwisampulu ethile.