Twabaye inararibonye mu gukora.Gutsindira ubwinshi mubyemezo byingenzi byisoko ryayo kubiciro Bigabanutse Ubushinwa Bwiyumvamo cyane Intambwe imwe Probe Rt-QpcrKit V2, Ubwiza ni uruganda 'ubuzima, Wibande kubakiriya' isoko niyo soko yo kubaho kwiterambere niterambere, Dukurikiza inyangamugayo n imyizerere myiza yo gukora, dutegereje kuza kwawe!
Twabaye inararibonye mu gukora.Gutsindira benshi mubyemezo byingenzi byisoko ryayoUbushinwa Taq ADN Polymerase, Qpcr, Isosiyete yacu isezeranya: ibiciro byumvikana, igihe gito cyo gukora na serivisi ishimishije nyuma yo kugurisha, turakwemera kandi gusura uruganda rwacu igihe cyose ubishakiye.Twifurije noneho kugira ubucuruzi bushimishije kandi burebure hamwe !!!

Ibisobanuro

Iki gikoresho gikoresha sisitemu idasanzwe ya lysis buffer ishobora kurekura byihuse RNA mumikorere yimikorere yimikorere ya RT-qPCR, bityo bikuraho inzira yo kweza RNA itwara igihe kandi ikora cyane.Inyandikorugero ya RNA irashobora kuboneka muminota 7 gusa.5 × Direct RT ivanze na 2 × Imiyoboro ya qPCR ivanze-SYBR itangwa nigitabo irashobora kwihuta kandi neza kubona ibisubizo nyabyo-byuzuye bya PCR.

5 × Direct RT ivanze na 2 × Direct qPCR ivanze-SYBR ifite kwihanganira imbaraga za inhibitor, kandi lysate yintangarugero irashobora gukoreshwa nkicyitegererezo cya RT-qPCR itaziguye.Iki gikoresho kirimo RNA idasanzwe-yuzuye ya Foregene reverse transcriptase, hamwe na polymerase ya D-Taq ishyushye, DNTPs, MgCl₂, reaction ya reaction, optimizer ya PCR na stabilisateur.

Ibisobanuro

200 × 20μl Rxns, 1000 × 20μl Rxns

Ibigize ibikoresho

Ibiranga ibyiza

■ Byoroshye kandi byiza: hamwe na tekinoroji ya Direct Direct RT, ingero za RNA zirashobora kuboneka muminota 7 gusa.

Icyitegererezo gikenewe ni gito, nkuko selile 10 zishobora kugeragezwa.

Thr Kwinjiza cyane: irashobora kumenya vuba RNA mu ngirabuzimafatizo zifite 384, 96, 24, 12, 6-amasahani meza.

Er ADN Eraser irashobora gukuraho vuba genome zasohotse, bikagabanya cyane ingaruka kubisubizo byakurikiyeho.

System Optimized RT na qPCR sisitemu ituma intambwe ebyiri RT-PCR ihinduranya transcript neza kandi PCR ikarushaho kuba nziza, kandi ikarwanya RT-qPCR inhibitor.

Porogaramu

Igipimo cyo gushyira mu bikorwa: ingirabuzimafatizo.

- RNA yarekuwe na sample lysis: ikoreshwa gusa kuri RT-qPCR inyandikorugero yiki gitabo.

- Igikoresho kirashobora gukoreshwa mubikorwa bikurikira: gusesengura imvugo ya gene, kugenzura ingaruka zo gucecekesha gene ya siRNA, gusuzuma ibiyobyabwenge, nibindi.

Igishushanyo

Ububiko hamwe nubuzima bwa Shelf

Igice cya I cy'iki gikoresho kigomba kubikwa kuri 4 ℃;Igice cya II kigomba kubikwa kuri -20 ℃.

Foregene Protease Plus II igomba kubikwa kuri 4 ℃, ntugahagarike kuri -20 ℃.

Reagent 2 × Direct qPCR Ivanga-SYBR igomba kubikwa kuri -20 ℃ mu mwijima;niba ikoreshwa kenshi, irashobora kandi kubikwa kuri 4 ℃ kubikwa igihe gito (koresha muminsi 10) .Twabaye inararibonye mubukora.Gutsindira benshi mubyemezo byingenzi byisoko ryayo kugirango bigabanuke Ubushinwa Bwinshi Kumva neza Intambwe imwe Probe Rt-Qpcr Kit V2, Ubwiza nubuzima bwuruganda, Kwibanda kubyo abakiriya bakeneye ni isoko yo kubaho kwiterambere niterambere, Dukurikiza inyangamugayo n imyizerere myiza yo kwizera, dutegereje kuza kwawe!
Kugabanuka cyaneUbushinwa Taq ADN Polymerase, Qpcr, Isosiyete yacu isezeranya: ibiciro byuzuye, igihe gito cyo gukora na serivisi ishimishije nyuma yo kugurisha, turakwemera kandi gusura uruganda rwacu igihe cyose ubishakiye.Twifurije noneho kugira ubucuruzi bushimishije kandi burebure hamwe !!!

QuickEasyTM Cell Direct RT-qPCR Kit -Taqman

Injangwe.No.DRT-01021/01022

Kuri selile itaziguye RT-qPCR ukoresheje ≤ 1000,000 selile

Kumenyekanisha ibicuruzwa

Iki gicuruzwa gikoresha sisitemu idasanzwe ya lysis kugirango irekure vuba RNA ingero zumuco zumuco kubitekerezo bya RT-qPCR, bivanaho uburyo bwo kweza RNA butwara igihe kandi bukora cyane, niminota 7 gusa kugirango ubone icyitegererezo cya RNA gisabwa, hamwe na 5 × Direct RT Mix, 2 × Direct qPCR Mix-Taqman yatanzwe nigitabo irashobora kubona vuba kandi neza ibisubizo byuzuye bya PCR.

5 × Direct RT ivanze na 2 × Direct qPCR Ivanga-Taqman ifite kwihanganira imbaraga za inhibitor kandi irashobora gukora neza kandi igahinduka ikoresheje lysate yicyitegererezo igomba gupimwa nkicyitegererezo.Reagent irimo Foregene Reverse Transcriptase, Hot D-Taq ADN Polymerase, DNTPs, MgCl₂, Buffer Reaction, PCR Optimizer na Stabilizer, irashobora gukoreshwa na lysis buffer kugirango byihuse kandi byoroshye kumenya ingero, kandi ifite ibiranga sensibilité yo hejuru, umwihariko kandi uhamye.

Ibiranga ibicuruzwa

Byoroheje, bikora neza Tekinoroji ya Cell Direct RT ifata iminota mike 7 kugirango ubone ingero za RNA.

Icyitegererezo gisabwa ni gito, kandi byibuze selile 10 zifite imico irashobora gukoreshwa mubigeragezo.

Ibicuruzwa byinshi kugirango RNA yihutire kubona ingirabuzimafatizo nka 384, 96, 24, 12, na 6-amariba.

ADN Eraser irashobora gukuraho vuba genome zasohotse, bikagabanya cyane ingaruka kubisubizo byakurikiyeho.

Sisitemu nziza ya RT na qPCR ituma intambwe ebyiri RT-PCR hamwe no kwandukura neza, kwihariye, hamwe no kwihanganira RT-qPCR.

Porogaramu

Igipimo cyo gusaba: Ingirabuzimafatizo.

Icyitegererezo lysis yasobanuye RNA: ikoreshwa gusa nkintambwe ebyiri RT-qPCR.

Ibikoresho birashobora gukoreshwa muburyo bukurikira: isesengura ryerekana imiterere ya gene, gupima allele, gusuzuma ibiyobyabwenge, nibindi.

Imipaka ntarengwa

Ibice byongerewe imbaraga ≤ 300 bp.

Ibikoresho bikoreshwa mumikorere mishya yumuco.

Kugenzura ubuziranenge bwibicuruzwa

Ukurikije uburyo bwo gucunga ubuziranenge bwa FOREGENE, buri cyiciro cyibikoresho bya selile Direct RT-qPCR bigeragezwa inshuro nyinshi kugirango hamenyekane ubwizerwe n’umutekano bya buri cyiciro cyibikoresho.

Ibirimo

*:Akagari ka Lysis, 5 × Direct RT ivanze, 2 × Direct qPCR ivanze-Taqman irashobora kugurwa ukwayo, ibisobanuro byatanzwe kumugereka wa 1 (URUPAPURO 13).

Imiterere yo kubika

1. Uburyo bwo kohereza

Inzira yose yubushyuhe buke bwo gupakira agasanduku ko gutwara, kugirango umenye neza ko ibikoresho biri muri <4 ° C.

2. Ububiko

Bika igice cya I kuri 4 ° C naho igice cya II kuri -20 ° C.

Foregene Protease Plus II igomba kubikwa kuri 4 ° C, ntikonje kuri -20 ° C.

Reagent 2 × Direct qPCR Ivanga-Taqman ibikwa kuri -20 ° C, cyangwa kuri 4 ° C kugirango ikoreshwe igihe gito iyo ikoreshejwe kenshi (muminsi 10).

Ibikoresho bigize ibikoresho

Buffer CL: Itanga ibidukikije bisabwa kugirango selile lysis reaction.

Buffer ST: Ihagarika ibintu bifatika muri lysate kugirango wirinde ingaruka kuri RT ikurikira.

Gusiba ADN: Gukuraho ADN, ingaruka zo gukuraho genome mubushakashatsi bwakurikiyeho.

5 × Direct RT ivanze: Irimo RNA ihanitse ya Foregene Reverse Transcriptase, RNase Inhibitor, dNTPs, stabilisateur, abongera imbaraga, optimizers, hamwe na primers transcription kugirango bahuze neza (Random Primer, Oligo (dT)₁₈Primer).

Foregene Protease Plus II: Mu rwego rwa lysis buffer, selile zatewe kugirango zirekure aside nucleic.

2.₂, reaction ya reaction, optimizer ya PCR, hamwe na stabilisateur.

20 × Irangi ryerekana ROX: Mubisanzwe bikoreshwa mubikoresho nyabyo bya PCR byongera imbaraga za ABI, Stratagene nandi masosiyete, bikoreshwa muguhindura itandukaniro riri hagati yigituba cya PCR nigituba cyatewe namakosa ya PCR.20 × ROX Reference Irangi ryibikoresho bisabwa kubikoresho bitandukanye biratandukanye, kandi uyikoresha arashobora kongeramo ukurikije icyifuzo cyibanze cyibikoresho.

Ubusa ddH₂O: RNase-idafite sterilize ultra amazi meza yintambwe ebyiri RT-qPCR.

Icyitonderwa:(Witondere gusoma neza witonze mbere yo gukoresha ibikoresho)

Witondere uburyo bwo gukora igerageza kugirango wirinde kwanduzanya hagati yintangarugero.

Witondere isuku yibidukikije bigeragezwa nibikoresho kugirango wirinde kwanduza RNase no kwangirika kwa RNA.

Fata ingirabuzimafatizo nshya cyangwa zabitswe neza kandi ntuzigere ukoresha ingero zifata inshuro nyinshi.

5 × Kuvanga qPCR itaziguye, 2 × Imvange ya qPCR-Taqman igomba kwirinda gukonjesha inshuro nyinshi, bitabaye ibyo bizagira ingaruka ku kwandukura no gukora neza kwa PCR.

PrepaIbiryombereimikorere

Witondere gusoma amabwiriza witonze mbere yo gukoresha iki gikoresho.Akagari ka Direct Direct RT-qPCR Kit biroroshye, byoroshye, kandi byihuse gukora, kandi amabwiriza atanga amakuru yuzuye kubyerekeye ibikoresho byose nuburyo bwo kubikoresha neza.Nyamuneka tegura ibikoresho nibikoresho byubushakashatsi bikenewe mbere yo kubikoresha.

Ibikoresho byubushakashatsi nibikoresho

Cell Ingirabuzimafatizo.

◆ 1.5 ml cyangwa ml 2, RNase- / DNase-Yubusa ya centrifuge, RNase- / DNase-Ubusa, 0.2 ml sterile qPCR.

Machine qPCR imashini, pipette, tabletop centrifuge (≥13.400×g) (ukurikije ibikenewe mu bushakashatsi), nibindi.

Umutekano

◆ Iki gicuruzwa kigamije ubushakashatsi bwa siyansi gusa, nyamuneka ntukoreshe imiti, ivuriro, ibiryo ndetse no kwisiga.

◆ Mugihe ukoresheje imiti, ambara imyenda ya laboratoire, gants, ibirahure birinda, nibindi.

IgikorwaKuyobora

Sisitemu ya Lysis selile, sisitemu ya RT, hamwe na qPCR ibisubizo byinyongera birashobora kugurwa ukundi, kubisobanuro birambuye kumugereka wa 1 (URUPAPURO 13).

Imiyoboro

Igisubizo: Icyitegererezo cya RNA kurekura

1.Ingirabuzimafatizo zabanjirijwe: Koza isahani yumuco wa selile hamwe na PBS ikonje, hanyuma utere selile (10-10⁶), 10⁶ kurenza umubare w'utugingo ngengabuzima, birasabwa Foregene Akagari RNA Yigunga Igikoresho Cyuzuye (DE-03111) cyangwa Igikoko Cyuzuye RNA Isolation Kit (DE-03011) kugirango ikuremo RNA.

1.1.Ingirabuzimafatizo (24- isahani nziza nkurugero)

1.1.1.Menya umubare w'utugingo ngengabuzima muri buri riba, menya ko umubare w'utugingo ngengabuzima ari 1 × 10⁵, kandi ukoreshe umuyoboro kugirango ukureho umuco wumuco mubiryo byumuco.

1.1.2.Ongeramo 200 μl zabanje gukonjeshwa 1 × PBS kuri buri riba.Ntugahindure inshuro nyinshi kandi ukure PBS kumariba.Shyira isahani hanyuma ukureho PBS nyinshi zishoboka.Komeza ku ntambwe ya 2.

1.1.3.Ibyokurya bitandukanye byumuco utugari cyangwa imbonerahamwe yerekana nimero 1-1 mubiryo byumuco w'akagari byongeweho mbere ya 1 × PBS yo koza selile.

Imbonerahamwe1-1: Igipimo cya PBS kumibare itandukanye ya selile

Icyitonderwa：Kugirango tumenye neza ingirabuzimafatizo，umubare munini wo gutakaza selile wirinze mugihe cyo gukaraba.

1.2.Utugingo ngengabuzima cyangwa ingirabuzimafatizo zifatika zifite amasahani adahwitse

1.2.1.Ingirabuzimafatizo zifatika zifite amasahani atari menshi (selile zo guhagarika zitangirira ku ntambwe ikurikira 1.2.2), gukusanya no gutandukanya selile ukurikije uburyo busanzwe bwo gukusanya selile, hanyuma ukabishyira mu isahani yumuco cyangwa umuyoboro wa centrifuge;niba trypsinisation ikoreshwa, bisaba centrifugation yo gukusanya selile no gukuraho trypsin isigaye, wongeyeho PBS yahagaritse selile muri selile zitandukanye kugirango ikwirakwize selile.

1.2.2.Nyuma yumubare wutugari tumaze kubarwa, selile selile 1 × 10⁵ imwe kugeza kuri centrifuge, gukusanya selile ukoresheje centrifugation kuri 1000 × g kuminota 10.

1.2.3.Ongeramo 200 μl PBS kumuyoboro wa centrifuge, ntukongere inshuro nyinshi, kandi wifuze neza PBS.komeza utere intambwe ya 2. (Niba bigoye kugwa kandi selile zongeye guhagarikwa, birashobora gukorwa 1000 × g centrifuged min 10 min nyuma yo guta ndengakamere, pellet selile ikomeza intambwe ya 2)

2.Cell lysis: Kuraho Buffer CL, ubushyuhe bwayo bungana nubushyuhe bwicyumba, ADN Eraser na Foregene Protease Plus II, ukurikije imbonerahamwe ikurikira 1-2 sisitemu ya lysis yateguwe: (Igisubizo cya Lysis cyiteguye gukoreshwa).

Imbonerahamwe1-2: clavage gutegura sisitemu (Icyitonderwa: mugutegura kurubura)

3..

Icyitonderwa：Kugirango wirinde kwibumbira hamwe, nyamuneka mugihe imiyoboro ya pipette yahinduwe kuri 200μl cyangwa munsi yayo.Ingirabuzimafatizo zishobora kugaragara nyuma ya lysis, nibisanzwe.

4..

Icyitonderwa：Umuyoboro wa pipette wajugunywe munsi yubuso, ukemeza ko lysate yongeyeho，kugirango wirinde kwibumbira hamwe, nyamuneka mugihe imiyoboro ya pipeti yahinduwe kuri 200μl cyangwa munsi yayo.

Imbonerahamwe 1-3：Ongeraho Buffer ST

5.Lysate ikoreshwa mubushakashatsi bwakurikiyeho RT-qPCR.Niba ubushakashatsi bwakurikiyeho budashobora gukorwa mugihe, nyamuneka ubigumane kurubura bitarenze 2h, hanyuma ubike kuri -20 ℃ cyangwa -80 ℃ (bitarenze amezi atatu).

B: Gutegura sisitemu ya RT

1.Kuramo 5 × Direct RT ivanze hanyuma uyishyire mubwogero bwa barafu, ureke bishonge bisanzwe, hanyuma ubivange witonze kugirango bikoreshwe nyuma;fata RNase-Yubusa ddH2O hanyuma uyishonge hanyuma uyishyire mubwogero bwa ice kugirango ukoreshwe nyuma.Tegura uburyo bwo kubyitwaramo kurubura ukurikije Imbonerahamwe 2-1 hepfo.

Imbonerahamwe 2-1: Gutegura sisitemu ya reaction ya RT

2.Nyuma yo kurangiza sisitemu, kuvanga buhoro buhoro hamwe na centrifuged muri make mumeza ikurikira 2 -2 reaction ya RT reaction.

Imbonerahamwe 2-2: Igenamiterere rya RT

3.Nyuma yo kurangiza reaction, ibicuruzwa byakozwe byashyizwe kumurongo kuri qPCR, nyamuneka shyira igihe kirekire -20 ℃ cyangwa -80 ℃.

Icyitonderwa: Bitewe no gukoresha inyandikorugero idasukuwe, imvura yera irashobora kugaragara mubicuruzwa byandukuwe.Ibi ni ibintu bisanzwe.Centrifuge ndengakamere ako kanya kubushakashatsi bwakurikiyeho.

Igisubizo cya RT cyakemuwe cyongewe kumurongo ukurikira Intambwe nyayo Igihe cya PCR reaction, birasabwa kongeramo amafaranga ari hagati ya 10-30% ya sisitemu yo kwitwara.

C: qPCR itegura sisitemu yo gutegura

1.Umubare ukwiye wa B wateguwe mu ntambwe ya cDNA ukurikije imbonerahamwe ikurikira 3-1 kugirango utegure sisitemu yo kwitwara.

Icyitonderwa: Ingano ya cDNA yerekana 10-30% ya sisitemu ya qPCR.Kurugero, muri sisitemu ya 20μl qPCR, ongeramo 2-6 μl ya lysis buffer, ariko ntibirenze 6 μl.

2.Gutezimbere ibintu byiza bya qPCR (Annealing ubushyuhe ， nibindi.) Kubisubizo bya qPCR (Imiterere yimyitwarire yatanzwe kumeza 3-2).

Icyitonderwa: Gerageza gukoresha ibintu byateganijwe kugirango qPCR ibone ibisubizo kugirango ubone ibisubizo byiza.

Imbonerahamwe 3-1: Gutegura sisitemu ya reaction ya PCR

1 *: Kwibanda kwa primer birashobora guhindurwa murwego rwa 50-900 nM mugihe imikorere ya primer reaction ari mibi.

Icyitonderwa: Sisitemu ya qPCR irashobora guhindurwa ukurikije ibikenewe mubigeragezo hamwe na fluorescence cycler.Kuri qPCR muri 50μsisitemu, hindura dosiye ya reagent ukurikije 20μSisitemu.

2 *: Hitamo icyerekezo cya nyuma cya ROX Reference Dye ukurikije fluorescence ingano yumuriro wumuriro.Ibikenewe cyane ROX Yerekana Irangi ryibara rya fluorescence yibisanzwe byamagare byerekanwe kumeza ikurikira:

Imbonerahamwe 3-2: qPCR ibisubizo byerekana

Icyitonderwa: Kugirango ubone ingaruka nziza za qPCR, icyiciro cya PCR kirashobora gukoreshwa mugutezimbere imiterere yimyitwarire inyandikorugero zitandukanye na primers zitandukanye.Imiterere ya PCR iratandukanye bitewe nisesengura rya fluorescence, inyandikorugero, primer, nibindi. Mubikorwa byihariye, uburyo bwiza bwo kubyitwaramo bugomba gutegurwa ukurikije imiterere yihariye ya fluorescence ingano yumuriro wumuriro, ubwoko bwicyitegererezo, ingano yikigice cyinyungu, urutonde rwibanze rwibice byongerewe hamwe nibirimo bya GC hamwe nuburebure bwa primers, harimo ubushyuhe bwa annealing, igihe cyo kubyitwaramo, nibindi.

Igihe nyacyo PCR primer igishushanyo mbonera

Imbere Primer na Reverse Primer

Kubihe Byukuri PCR, igishushanyo mbonera ni ngombwa cyane.Ibanze bifitanye isano nuburyo bwihariye bwo kongera PCR, kandi birashobora gushushanywa hifashishijwe amahame akurikira:

Long Uburebure bwa primer: 18-30bp.

Content Ibirimo GC: 40-60%.

Value Tm agaciro: Porogaramu ishushanya, nka Primer 5, irashobora gutanga Tm agaciro ka primer.Indangagaciro za Tm zo hejuru no kumanuka primers zigomba kuba hafi bishoboka.Inzira yo kubara ya Tm irashobora kandi gukoreshwa: Tm = 4 ° C (G + C) + 2 ° C (A + T).Iyo ukora PCR, ubushyuhe buri munsi ya primer Tm agaciro ka 5 ° C muri rusange byatoranijwe nkubushyuhe bwa annealing (kwiyongera gukwiranye nubushyuhe bwa annealing birashobora kongera umwihariko wa reaction ya PCR).

Ibicuruzwa nibicuruzwa bya PCR:

◆ Gushushanya primer PCR yongerera ibicuruzwa uburebure nibyiza 100-150bp.

◆ Gushushanya primers mugice cya kabiri cyubatswe cyicyitegererezo kigomba kwirindwa bishoboka.

Irinde gushiraho ibice 2 cyangwa byinshi byuzuzanya hagati ya 3 ′ impera yimbere na epfo na ruguru.

◆ Primer 3 base base base ntishobora kuboneka hamwe na 3 yongeyeho G cyangwa C.

◆ Primers ubwayo ntishobora kugira ibyuzuzanya, bitabaye ibyo hazashyirwaho imiterere yimisatsi, bigira ingaruka kuri PCR.

◆ ATCG igomba gukwirakwizwa uko bishoboka kose murwego rwa primer, kandi 3 ′ base base igomba kwirindwa nka T.

Umugereka1：CellRT-qPCR Kit component

1.Cell Lysis Igisubizo