Nke ahụ nwere àgwà ziri ezi na nke na-aga n'ihu maka mmasị ndị ahịa, nzukọ anyị na-eme ka ngwaahịa anyị dịkwuo mma iji mejuo ọchịchọ nke ndị na-azụ ahịa na n'ihu na-elekwasị anya na nchekwa, ntụkwasị obi, nkọwa gburugburu ebe obibi, na ihe ọhụrụ nke Ụlọ ọrụ Supply China Convenient and Quick Rt-PCR Dspathtm 4X One-Step Multiplex Master Mix, Anyị ahụmahụ mgbagwoju anya workforce nwere ike na-akwado gị dum.Anyị ji obi ụtọ na-anabata gị ka ị kwụsị site na webụsaịtị anyị na ụlọ ọrụ ma zigara anyị ajụjụ gị.
Nke ahụ nwere àgwà ziri ezi na nke na-aga n'ihu maka mmasị ndị ahịa, nzukọ anyị na-eme ka ngwaahịa anyị dịkwuo mma iji gboo ihe ndị na-azụ ahịa na-achọ ma na-elekwasị anya na nchekwa, ntụkwasị obi, nkọwa gburugburu ebe obibi, na mmepụta ọhụrụ nkeChina Taq DNA Polymerase, Qpcr, Anyị nwere ugbu a ihe ngwọta kasị mma na nkà ahịa na nkà na ụzụ team.Na mmepe nke anyị ụlọ ọrụ, anyị na-enwe ike na-enye ndị ahịa kasị mma ngwaahịa, ezi oru nkwado, zuru okè mgbe-sales ọrụ.

Nkọwa

Ngwa a na-eji sistemu nchekwa lysis pụrụ iche nke nwere ike hapụ RNA ngwa ngwa site na nlele cell emebere maka mmeghachi omume RT-qPCR, si otú a na-ewepụ usoro ịdị ọcha nke RNA na-ewe ma na-arụsi ọrụ ike.Enwere ike nweta ndebiri RNA n'ime naanị nkeji 7.Ngwakọta 5 × Direct RT Mix na 2 × Direct qPCR Mix-SYBR reagents nke ngwa ahụ na-enye nwere ike nweta nsonaazụ PCR ozugbo na nke ọma.

5 × Direct RT Mix na 2 × Direct qPCR Mix-SYBR nwere nnabata inhibitor siri ike, enwere ike iji lysate nke ihe nlele ahụ dị ka ndebiri maka RT-qPCR ozugbo.Ngwa a nwere RNA pụrụ iche Foregene reverse transcriptase, yana Hot D-Taq DNA polymerase, dNTPs, MgCl.₂, ihe nchekwa mmeghachi omume, PCR optimizer na stabilizer.

Nkọwapụta

200 × 20μl Rxns, 1000 × 20μl Rxns

Ngwa ngwa

Atụmatụ & uru

∎ Dị mfe ma dị irè: site na teknụzụ Cell Direct RT, enwere ike nweta ihe nlele RNA n'ime naanị nkeji asaa.

■ Ọchịchọ nlele dị ntakịrị, ebe ọ bụ na enwere ike ịnwale mkpụrụ ndụ 10.

∎ Mgbakwunye dị elu: ọ nwere ike ịchọpụta RNA ngwa ngwa n'ime sel ndị emebere na 384, 96, 24, 12, 6-well plates.

∎ DNA Eraser nwere ike wepụ ngwa ngwa genomes ewepụtara, belata mmetụta dị na nsonaazụ nnwale na-esote.

∎ Sistemu RT na qPCR emezigharịrị na-eme ka ntụgharị ntụgharị RT-PCR nzọụkwụ abụọ rụọ ọrụ nke ọma yana PCR akọwapụtara nke ọma, ma na-eguzogide ndị na-egbochi mmeghachi omume RT-qPCR.

Ngwa ngwa

Oke ngwa: mkpụrụ ndụ omenala.

- RNA wepụtara site na nlele lysis: naanị ọdabara na ndebiri RT-qPCR nke ngwa a.

- Enwere ike iji ngwa ahụ maka ebumnuche ndị a: nyocha okwu mkpụrụ ndụ ihe nketa, nkwenye nke mmetụta ịgbachitere mkpụrụ ndụ ihe nke siRNA, nyocha ọgwụ, wdg.

Eserese

Nchekwa na Shelf ndụ

A ga-echekwa akụkụ nke mbụ nke ngwa a na 4 ℃;Ekwesịrị ịchekwa akụkụ II na -20 ℃.

Ekwesịrị ịchekwa Foregene Protease Plus II na 4 ℃, echekwala na -20 ℃.

Ekwesịrị ịchekwa Reagent 2 × Direct qPCR Mix-SYBR na -20℃ n'ọchịchịrị;ọ bụrụ na-eji ugboro ugboro, ọ nwekwara ike na-echekwara na 4 ℃ maka obere oge nchekwa (jiri elu n'ime 10 ụbọchị) .Nke nwere a nti na-aga n'ihu àgwà nke ahịa mmasị, anyị nzukọ anọgide mma anyị ngwaahịa àgwà na-egbo ndị na-azụ ahịa na n'ihu na-elekwasị anya na nchekwa, ntụkwasị obi, gburugburu ebe obibi nkọwa, na ọhụrụ nke Factory Supply China Convenient na Quick Rt-Pemce Multi-Part DCRD anyị nwere ike bụrụ mgbagwoju anya. jiri obi dum na-akwado gị.Anyị ji obi ụtọ na-anabata gị ka ị kwụsị site na webụsaịtị anyị na ụlọ ọrụ ma zigara anyị ajụjụ gị.
Nkwanye ụlọ ọrụChina Taq DNA Polymerase, Qpcr, Anyị nwere ugbu a ihe ngwọta kasị mma na nkà ahịa na nkà na ụzụ team.Na mmepe nke anyị ụlọ ọrụ, anyị na-enwe ike na-enye ndị ahịa kasị mma ngwaahịa, ezi oru nkwado, zuru okè mgbe-sales ọrụ.

Real Time PCR primer ụkpụrụ imewe

Na-aga n'ihu Primer na Reverse Primer

Maka Real Time PCR, imewe primer dị ezigbo mkpa.Primers metụtara nkọwapụta na arụmọrụ nke nkwalite PCR, enwere ike hazie ya na ntụnyere ụkpụrụ ndị a:

Ogologo oge mbụ: 18-30bp.

Ọdịnaya GC: 40-60%.

Uru Tm: Akụrụngwa imewe nke mbụ, dị ka Primer 5, nwere ike inye uru Tm nke primer.Ụkpụrụ Tm nke ndị na-ebuli elu na nke mgbada kwesịrị ịdị nso dịka o kwere mee.A nwekwara ike iji usoro mgbako Tm: Tm = 4 °C (G + C) + 2 Celsius C (A + T).Mgbe ị na-arụ PCR, a na-ahọrọ okpomọkụ dị n'okpuru uru Tm primer nke 5 Celsius dị ka okpomọkụ na-ekpo ọkụ (mmụba kwekọrọ na okpomọkụ na-ekpo ọkụ nwere ike ịbawanye nkọwa nke mmeghachi omume PCR).

Ngwaahịa mbụ na PCR:

Design primer PCR amplification ogologo ngwaahịa bụ ọkachamma 100-150bp.

Ekwesịrị ịzere primers imewe na mpaghara nhazi nke abụọ nke ndebiri dị ka o kwere mee.

Zenarị nhazi ntọala 2 ma ọ bụ karịa n'etiti nsọtụ 3 nke elu na ala ala.

Primer 3′ terminal base enweghị ike ịnọ ya na 3 mgbakwunye G ma ọ bụ C.

Ndị primers n'onwe ha enweghị ike ịnwe ihe mgbakwunye, ma ọ bụghị ya, a ga-emepụta usoro ntutu isi, na-emetụta mmụba PCR.

Ekwesịrị kesaa ATCG n'otu n'otu dịka o kwere mee n'usoro nke mbụ, yana ebe njedebe 3′ kwesịrị izere ka T.

Ihe mgbakwunye 1: Mkpọ mgbakwunye akụrụngwa Direct RT-qPCR Kit

1.Cell Lysis Solution