I-FastKing gDNA Ukususa i-RT SuperMix

I-A-1 RNA yehlisiwe

——Qondisa izinga eliphezulu le-RNA ngaphandle kokungcola. Izinto okukhishwa kuzo i-RNA kufanele zibe zintsha ngangokunokwenzeka ukuvimbela ukonakala kwe-RNA. Hlaziya ubuqotho be-RNA ku-gel esetshenzisiwe ngaphambi kokuphendula kwe-RT. Ngemuva kokukhishwa kwe-RNA, kufanele igcinwe ku-100% formamide. Uma kusetshenziswa i-RNase inhibitor, ithempelesha yokushisa kufanele ibe ngu- <45 ° C, kanti i-pH kufanele ibe ngaphansi kuka-8.0, uma kungenjalo i-inhibitor izokhipha yonke i-RNase eboshiwe. Ngaphezu kwalokho, i-RNase inhibitor kufanele ingezwe kuzixazululo eziqukethe ≥ 0.8 mM DTT.

I-A-2 RNA iqukethe ama-inhibitors we-reverse transcript reaction

——Reverse transcription inhibitors ifaka i-SDS, i-EDTA, i-glycerol, i-sodium pyrophosphate, i-spermidine, i-formamide, i-guanidine usawoti, njll. Hlanganisa i-RNA yokulawula nesampuli, bese uqhathanisa isivuno nempendulo ye-RNA yokulawula ukuze uhlole ukuthi ingabe ikhona yini i-inhibitor. Geza imvula ye-RNA nge-70% (v / v) ethanol ukususa ama-inhibitors.

I-A-3 annealing eyanele yama-primers asetshenziselwa ukuhlanganisa umucu wokuqala we-cDNA

- Nquma ukuthi i-annealing lokushisa kufanelekile kuma-primers asetshenziswe ekuhlolweni. Ngama-hexamers angahleliwe, kunconywa ukuthi ugcine izinga lokushisa liku-25 ° C ngemizuzu eyi-10 ngaphambi kokufinyelela izinga lokushisa lokuphendula. Kuma-primers aqondene nezakhi zofuzo (GSP), zama enye i-GSP, noma ushintshele ku-oligo (dT) noma i-hexamer engahleliwe.

Inani elincane le-A-4 lokuqala i-RNA

- Khulisa inani le-RNA. Ngamasampula e-RNA angaphansi kuka-50 ng, i-0.1 μg kuya ku-0.5 μg i-acetyl BSA ingasetshenziswa ekuhlanganisweni kokuqala kwe-strand cDNA

A-5 Ukulandelana okuqondiwe akuboniswa kuzicubu ezihlaziyiwe.

——Zama ezinye izicubu.

Ukusabela kwe-A-6 PCR kwehluleka

——Kwezinyathelo ezimbili i-RT-PCR, ithempulethi ye-cDNA esinyathelweni se-PCR ayikwazi ukudlula i-1/5 yevolumu yokuphendula.

Ukuncishiswa okungacacisiwe kwama-primers nama-templates

—— Ukuphela kuka-3'kokuqala kwama-primers akufanele kube ne-2-3 dG noma i-dC. Sebenzisa ama-primers akhethekile we-Gene ku-strand synthesis yokuqala esikhundleni sama-primers angahleliwe noma i-oligo (dT). Sebenzisa ukushisa okuphezulu kokuncisha emijikelezweni embalwa yokuqala, bese izinga lokushisa elingaphansi elincishisiwe. Sebenzisa i-hot-start Taq DNA polymerase ye-PCR ukuthuthukisa ukucaciswa kokuphendula.

Idizayini engalungile ye-A-2 yama-primers aqondene nezakhi zofuzo

- Landela imigomo efanayo yokuklama i-amplification primer design.

I-A-3 RNA ingcoliswe yi-genomic DNA

—Thola i-RNA nge-PCR-grade DNase I. Setha ukusabela kokulawula ngaphandle kokuloba okubuyela emuva ukuthola ukungcola kwe-DNA.

Ukwakhiwa kwe-primer dimer

Ama-primers okuklama ngaphandle kokulandelana okuhambisanayo ekugcineni kuka-3.

A-5 Mg ephakeme kakhulu²⁺ ukuhlushwa

—— Lungiselela uMg²⁺ ukugxila kuthempulethi ngayinye nokuhlanganiswa kokuqala

I-A-6 Ingcoliswe nge-DNA yangaphandle

—— Sebenzisa amathiphu amelana ne-aerosol nama-enzyme e-UDG.

A-1 Okuqukethwe komkhiqizo we-strand wokuqala kuphezulu kakhulu

- - Nciphisa inani lomkhiqizo wokuqala we-strand kusinyathelo esivamile se-PCR reaction.

Inani elingu-A-2 eliphakeme kakhulu ekuphenduleni kwe-PCR

- - Nciphisa okokufaka kokuqala.

A-3 Imijikelezo eminingi kakhulu

Lungiselela izimo zokuphendula ze-PCR futhi unciphise inombolo yomjikelezo we-PCR.

A-4 Ukushisa okuphansi kakhulu

—Ukwandisa izinga lokushisa lokunciphisela ukuvimbela ukuqalisa nokunweba okungacacisiwe.

I-A-5 Ukukhulisa okungacaciswanga kwezingcezu ze-oligonucleotide ezikhiqizwe ukonakala kwe-DNase kwe-DNA —— Khipha i-RNA esezingeni eliphezulu ukuvimbela ukungcoliswa kwe-DNA.

I-RT-PCR ukubuyisela emuva ukubhala phansi i-RNA ibe yi-cDNA, bese usebenzisa i-cDNA ebhaliwe ebuyiselwe njengesifanekiso sokuphendula kwe-PCR ukukhulisa isiqeshana esiqondisiwe. Khetha ama-primers angahleliwe, i-Oligo dT kanye nama-primers aqondene nezakhi zofuzo ngokuya ngezimo ezithile zesilingo. Onke ama-primers angenhla angasetshenziselwa i-eukaryotic cell mRNA ngaphandle kwesakhiwo se-hairpin.

I-Random primer: Ifanele i-RNA ende enesakhiwo se-hairpin, kanye nazo zonke izinhlobo ze-RNA ezinjenge-rRNA, mRNA, tRNA, njll. Zisetshenziselwa ukuphendula kwe-RT-PCR kwesifanekiso esisodwa.

I-Oligo dT: Ifanele i-RNA ene-PolyA tailing (i-prokaryotic RNA, i-eukaryotic i-Oligo dT rRNA ne-tRNA ayinayo imisila ye-PolyA). Ngoba i-Oligo dT iboshelwe kumsila wePolyA, ikhwalithi yamasampuli e-RNA iyadingeka ukuthi ibe phezulu, futhi noma inani elincanyana lokwehlisa izinga lizokwehlisa kakhulu inani lokuhlanganiswa okugcwele kwe-cDNA.

I-primer ekhethekile ye-Gene: Ihambisana nokulandelana kwethempulethi, efaneleke ezimeni lapho ukulandelana kwempokophelo kwaziwa.

Kunezindlela ezimbili:

1. Indlela yereferensi yangaphakathi: Ngokombono, i-cDNA izingcezu ze-DNA zobude obuhlukile, ngakho-ke umphumela we-electrophoresis yi-smear. Uma ubuningi be-RNA buphansi, awukho umkhiqizo ozobonakala ku-electrophoresis, kepha lokhu akusho ukuthi awukho umkhiqizo ozokwandiswa yi-PCR. Ngokuvamile, ireferensi yangaphakathi ingasetshenziselwa ukuthola i-cDNA. Uma ireferensi yangaphakathi inemiphumela, ikhwalithi ye-cDNA ingaqinisekiswa ngokuyisisekelo (ezimweni ezimbalwa, uma isiqeshana sofuzo esihlosiwe side kakhulu, kungahle kube nokuhlukile).

2. Uma kukhona ufuzo olwaziwe olwandiswa yilesi sifanekiso, lungaqinisekiswa ngabokuqala balesi sakhi. Ukukhuliswa kwesethenjwa sangaphakathi akusho ukuthi ayikho inkinga nge-cDNA. Ngoba ireferensi yangaphakathi inenqwaba ye-cDNA, kulula ukuyikhulisa. Uma i-cDNA yehliswa ngokwengxenye ngenxa yezizathu ezahlukahlukene, ngokubuka kokungenzeka, imiphumela ye-PCR yezakhi zofuzo eziqondiwe ngobuningi izothinteka kakhulu. Ngenkathi ireferensi yangaphakathi isaphezulu ngobuningi, ukukhulisa amandla kungenzeka kungathinteki.

Ukuwohloka kancane kwe-RNA. Thola ubuqotho bese uhlanza i-RNA

Okuqukethwe kwe-RNA yezinhlobo ezahlukahlukene kungahluka, kepha ngokujwayelekile, inani eliphelele le-RNA kufanele libe namabhande amabili acacile we-28S kanye ne-18S ku-gel electrophoresis, futhi ukukhanya kwebhande langaphambili kufanele kuphakame ngokuphindwe kabili kunokwalokhu okugcina. Ibhendi le-5S likhombisa ukuthi i-RNA yehlisiwe, futhi ukukhanya kwayo kuyalingana nezinga lokwehla. Ukukhuliswa ngempumelelo kwesethenjwa sangaphakathi akusho ukuthi ayikho inkinga nge-RNA, ngoba ireferensi yangaphakathi inenqwaba ephezulu, i-RNA ingakhuliswa inqobo nje uma ukonakala kungabi kubi. I-OD₂₆₀/ OD₂₈₀isilinganiso se-RNA emsulwa esilinganiswe nge-spectrophotometer kufanele sibe phakathi kuka-1.9 no-2.1. Inani elincane lokungcola kwamaprotheni ku-RNA kuzonciphisa isilinganiso. Uma nje inani lingaphansi kakhulu, i-RT ngeke ithinteke. Okubaluleke kakhulu kwi-RT ubuqotho be-RNA.

Ukunwetshwa kofuzo lwesithenjwa sangaphakathi kungakhombisa kuphela ukuthi i-RT iphumelele, kepha akuhlobene neze nekhwalithi yomucu we-cDNA. Ngoba izingcezu zezethenjwa zangaphakathi ngokuvamile zincane ngosayizi futhi ziveza okukhulu, kulula ukuphumelela ekubhaleni okuphindayo. Kodwa-ke, ubukhulu nokuvezwa kohlobo oluhlosiwe kuyahlukahluka kuye ngezakhi zofuzo. Ikhwalithi ye-cDNA ayikwazi ukwahlulelwa kuphela ngesethenjwa sangaphakathi ikakhulukazi izingcezu ezihlosiwe ezinde kune-2 kb.

Amanye amasampula anezakhiwo eziyinkimbinkimbi zesibili, noma anokuqukethwe okunothile kwe-GC, noma ayigugu ngobuningi obuphansi. Kulezi zimo, kufanele kukhethwe i-reverse transcriptase efanele ngosayizi wocezu lwenhloso nesampula. Okwezifanekiso ze-RNA ezinokuqukethwe okuphezulu kwe-GC nokwakheka okuyinkimbinkimbi kwesekondari, kunzima ukuvula isakhiwo sesibili emazingeni aphansi okushisa, noma nge-reverse transcriptase ejwayelekile. Kula mathempulethi, i-Quant Reverse Transcriptase ingakhethwa, ngoba ukusebenza kwayo okuphambene nomqondo ngokusobala kungcono kunalokho kwe-M-MLV series reverse transcriptase, engaguqula ukubhalwa kwamathempulethi ahlukahlukene e-RNA kahle futhi ibhale i-RNA ibe yi-cDNA strand yokuqala ize ifike ezingeni eliphezulu. Lapho usebenzisa i-reverse reverse transcriptase kit, uhlelo lwama-20 μl lungaguqula ngempumelelo kuphela ukubhala okungu-1 μg we-RNA ephelele. Sicela unake umthamo omkhulu we-RT wekithi. Uma ithempulethi ingezwe ngokweqile, ukuloba okuphindayo kuzokwenzela i-RNA ngobuningi obuphezulu. Ngakho-ke, kungcono ukuthi ungadluli umthamo omkhulu wesistimu.

A-1 Thola ukuthi i-RNA yehliswe isithunzi kakhulu nokuthi i-RT iphumelele yini

Ngokuvamile, isizathu sokwehluleka kokukhuliswa kwereferensi yangaphakathi kuvame ukubangelwa ukwehla okukhulu kwe-RNA. Esinye isizathu esingaba khona ukwehluleka kokuloba okuphindayo. Isethenjwa sangaphakathi asinakusetshenziswa njengezinga lokwahlulela ikhwalithi ye-cDNA single strand, kepha ingasetshenziswa njengezinga elijwayelekile lokwahlulela ukuthi ukuloba okuphindayo kuyaphumelela uma ingekho inkinga yekhwalithi ye-RNA. Into ebaluleke kakhulu kunqubo yokubhala okuphindayo ukugcina ukushisa okungaguquguquki kanye nohlelo lokuphendula njalo ukuze kuthuthukiswe ukusebenza kahle kokuphendula.

I-A-2 Nquma ukuthi ingabe iziqalo zokukhulisa izakhi zofuzo zereferensi yangaphakathi zinokwethenjelwa futhi uma kunezinkinga ngama-reagents asetshenziswa ku-PCR.

Ukuze kwenziwe i-quantification ehlobene, i-RNA kufanele ibalwe ngaphambi kokubhalwa phansi okuphindwayo, okudingekayo nakuma-kits amaningi wokubhala okuphindayo, ngokwesibonelo, ulinganise okokufaka kwe-RNA njenge-1 μg. Njengoba i-cDNA ebhalwe phansi iyisixazululo esihlanganisiwe, kufaka phakathi i-RNA, i-oligo dT, i-enzyme, i-dNTP, kanye nensalela encane ye-DNA, ukuphambuka kuzobangelwa, ngakho-ke akunakwenzeka ukukala ngokunembile i-cDNA. Ngakho-ke, i-quantification ye-RNA iyadingeka. Uma kubhekwa ukusebenza kwe-transcript okuphindayo kuyafana phakathi kwamasampuli ahlukahlukene, inani le-cDNA elitholakele kufanele lifane, futhi ukuhlaziywa kwenani kungakhombisa ukuqhathaniswa kwamazinga okuveza izakhi zofuzo ezahlukahlukene ngenani elifanayo le-RNA ephelele. Lapho wenza i-PCR yokulinganisa i-fluorescence ehambisanayo, i-cDNA yokulinganisa kungenzeka ingadingeki ngemuva kokubhalwa okuphindayo ngoba isakhi sangaphakathi senkomba singenziwa njengesethenjwa.

Ihlobene kakhulu nezakhi zofuzo, futhi ukubhalwa phansi kwesiqeshana eside akunakwenzeka ezinhlotsheni eziningi zofuzo. Okokuqala, ukusebenza kahle kokuloba okuphindayo kuncane kakhulu kunokwe-PCR. Okwesibili, indawo ecebile ye-GC nokwakheka kwesibili kwezakhi zofuzo eziningi kuvimbela kokubili ukubhalwa phansi kwe-reverse ne-PCR. Ekugcineni, ukuthembeka nokukhulisa ukusebenza kwe-PCR kunzima ukukuqinisekisa ngasikhathi sinye. Ngenqubo yokubhalwa phansi okuphindayo, akekho umuntu ongaqinisekisa ukuthi uthola izingcezwana ezinde zezakhi zofuzo eziphansi, ikakhulukazi usebenzisa i-oligo dT. Ngokuqondene ne-5 'UTR ene-GC eningi, kunzima kakhulu. Ngakho-ke, kuseseyindlela enengqondo yokuhlehlisa okulotshiweyo ngama-primers angahleliwe, thola amasayithi wemvelo wokuqhekeka esiqeshini esihlosiwe, ukhulise ngamasegmenti, bese wenza ukuvinjelwa kokugaywa nokuhlanganiswa. Ngokuvamile, kunzima ukukhulisa ngqo izingcezu ezinkulu kune-2 kb, kepha akunakwenzeka ngaso sonke isikhathi ukuthola: 1. Okokuqala, qinisekisa ubuqotho be-RNA / mRNA, nokukhethwa kwe-TRIZOL. I-2.M-MLV RT-PCR kit ingasetshenziswa ngqo. Nweba isikhathi sokuncipha futhi ukhulise inombolo yomjikelezo kunqubo yokukhulisa kahle. Ngenye indlela, i-PCR ehlanganisiwe ingasetshenziswa, noma yenze ukuphendula okukodwa noma okubili kuqala ngokunwetshwa ngendlela efanele nesikhathi sokunweba ngaphambi kokukhulisa okujwayelekile kwe-PCR, okungasiza ukunweba izingcezwana. Nakani ukwethembeka kwe-polymerase. I-3.Long Taq ingasetshenziswa ku-PCR ukuthola imiphumela emihle. Ukusetshenziswa kwesicelo sokuveza amaprotheni, kufanele kusetshenziswe ukuthembeka okuphezulu kwe-polymerase.

Kunezinhlobo ezimbili ze-reverse transcriptase enikezwa yi-TIANGEN: I-Quant / King RTase ne-TIANScript M-MLV. Umehluko omkhulu phakathi kwazo inani lokufaka lezifanekiso. I-Quant iyi-reverse transcriptase eyingqayizivele, eyehlukile kune-M-MLV esetshenziswa kakhulu etholakala ku-Moloney murine leukemia virus. I-Quant yi-high transactionase ephezulu esebenza kahle kakhulu evezwe ngokucophelela ngobunjiniyela i-Escherichia coli. I-Quant ilungele ukukhulisa ama-50 ng-2 μg we-RNA ngomsebenzi ophindaphindwayo wokubhala phansi kanye nesivuno esikhulu. Uma kuqhathaniswa ne-MMLV ejwayelekile noma i-AMV, isici esikhulu kakhulu se-Quant ukuthi inobuhlobo obuqinile nezifanekiso ze-RNA futhi ingaguqula amathempulethi ayinkimbinkimbi ngokubhaliwe ngaphandle kokushiswa okuphezulu kokushisa. Okwezifanekiso ezinokuqukethwe okuphezulu kwe-GC, ukusebenza ngempumelelo okuphakeme kuphezulu. Kodwa-ke, le reverse transcriptase inomsebenzi we-RNase H, ongathinta ubude bomkhiqizo we-cDNA (ofanele amathempulethi angu- <4.5 kb). Ngokuloba okujwayelekile okubuyela emuva, Kunconywa i-TIANScript MMLV reverse transcriptase. Le RTase iyi-enzyme eguquliwe enomsebenzi obuthakathaka kakhulu we-RNase H, efanelekile ukuhlanganiswa kwama-cDNA amade (> 5 kb) cDNA.

Isinyathelo esisodwa sokuphindisela emuva nokukhuliswa kwe-PCR kuqedwa kushubhu efanayo ngaphandle kokuvula ikhava yeshubhu phakathi kokuhlanganiswa kwe-cDNA nokukhulisa, okusiza ukunciphisa ukungcoliswa. Njengoba wonke amasampula e-cDNA atholakalayo asetshenziselwa ukukhulisa, ukuzwela kuphakeme, ubuncane be-0.01 pg ye-RNA ephelele. Kwi-RTPCR yesinyathelo esisodwa esiphumelelayo, ama-primers aqondene nezakhi zofuzo ngokuvamile asetshenziselwa ukuqala ukuhlanganiswa kwe-cDNA. Indlela enezinyathelo ezimbili, okuyi-reverse transcript ne-PCR amplification yenziwa ngezinyathelo ezimbili. Okokuqala ukuloba okubuyela emuva kwenziwa kusuka kuthempulethi ye-RNA ukuthola i-cDNA, futhi i-cDNA etholakele ibhekene nokuphendula okukodwa noma okuningi kwe-PCR. Indlela enezinyathelo ezimbili ingasebenzisa i-oligo (dT) noma ama-primers angahleliwe ukuqondisa ukuhlanganiswa kwe-strand yokuqala ye-cDNA, futhi ingaguqula ukuloba lonke ulwazi lwe-mRNA kusuka kusampula ethile.