TIANSeq RNA Frag/cDNA Synthesis Module

Tam sim no, kev siv tshuab thev naus laus zis siab yog feem ntau yog ua raws cov thev naus laus zis tom ntej. Raws li kev nyeem ntawv ntev ntawm cov thev naus laus zis sib txuas tom ntej muaj qhov txwv, peb yuav tsum ua txhaum tag nrho cov ntu ua ntu ntev mus rau hauv cov tsev qiv ntawv me me ua ntu zus. Raws li qhov xav tau ntawm kev sim ua ntu sib txawv, peb feem ntau xaiv ib leeg-ua ntu lossis ob-ntu ua ntu zus. Tam sim no cov DNA tawg ntawm cov tsev qiv ntawv sib txuas tom ntej no feem ntau raug faib hauv thaj tsam ntawm 200-800 bp.

a) DNA tsis zoo thiab muaj cov tshuaj tiv thaiv kab mob. Siv cov qauv DNA zoo los tiv thaiv kev ua haujlwm ntawm enzyme.

b) Tus nqi ntawm cov qauv DNA tsis txaus thaum siv PCR-tsis muaj txoj hauv kev los tsim cov tsev qiv ntawv DNA. Thaum cov tswv yim ntawm cov DNA tawg ua ntau dua 50 ng, PCR-tsis ua haujlwm tuaj yeem xaiv tau ua thaum lub sijhawm tsim cov tsev qiv ntawv. Yog tias daim ntawv theej ntawm lub tsev qiv ntawv qis dhau los ua ntu ntu ncaj qha, lub tsev qiv ntawv DNA tuaj yeem nthuav dav los ntawm PCR tom qab hloov kho qhov txuas.

c) RNA kev sib kis ua rau tsis raug tus lej DNA thawj zaug RNA kev sib kis tuaj yeem muaj nyob hauv cov txheej txheem kev ua kom huv ntawm cov genomic DNA, uas tuaj yeem ua rau DNA suav tsis raug thiab tsis txaus DNA thauj khoom thaum tsim tsev qiv ntawv. RNA tuaj yeem tshem tawm los ntawm kev kho nrog RNase.

A-1

a) Cov khoom me me (60 bp-120 bp) zoo li cov khoom me me feem ntau yog cov khoom tawg lossis cov dimers tsim los ntawm cov hloov pauv. Kev ua kom huv nrog Agencourt AMPure XP sib nqus hlaws dai tuaj yeem tshem tawm cov khoom tawg no kom zoo thiab ua kom muaj kev sib dhos zoo.

b) Cov tawg loj tshwm nyob rau hauv lub tsev qiv ntawv tom qab PCR kev nthuav dav Qhov loj ntawm lub tsev qiv ntawv DNA tawg ua ke yuav nce ntxiv 120 bp tom qab lub adapter txuas nrog. Yog tias DNA qhov tawg tau nce ntau dua 120 bp tom qab hloov kho qhov txuas, nws yuav tshwm sim los ntawm qhov txawv txav ntawm kev faib tawm ntawm PCR ntau dhau. Txo cov naj npawb ntawm PCR mus los tuaj yeem tiv thaiv qhov xwm txheej.

c) Qhov txawv me me ntawm cov tsev qiv ntawv DNA tawg tom qab hloov kho qhov ntev Qhov hloov pauv hauv cov khoom siv no yog 60 bp. Thaum ob qhov kawg ntawm qhov tawg tau sib txuas rau cov hloov pauv, qhov ntev yuav tsuas yog nce ntxiv 120 bp. Thaum siv lub adapter tsis yog qhov uas tau muab los ntawm cov khoom siv no, thov hu rau tus muag khoom kom muab cov ntaub ntawv cuam tshuam xws li qhov ntev adapter. Thov kom ntseeg tau tias kev sim ua haujlwm thiab ua haujlwm ua raws cov kauj ruam tau piav qhia hauv phau ntawv qhia.

d) Qhov txawv me me ntawm DNA qhov loj me ua ntej hloov pauv adapter Vim li cas rau qhov teeb meem no tuaj yeem tshwm sim los ntawm cov xwm txheej tsis raug thaum lub sijhawm DNA tawg. Lub sijhawm sib txawv ntawm cov tshuaj tiv thaiv yuav tsum tau siv rau kev nkag DNA sib txawv. Yog tias DNA nkag mus ntau dua 10 ng, peb pom zoo kom xaiv lub sijhawm tshuaj tiv thaiv ntawm 12 min raws li lub sijhawm pib rau kev ua kom zoo dua, thiab qhov tawg me me uas ua rau lub sijhawm no feem ntau yog nyob ntawm 300-500 bp. Cov neeg siv tuaj yeem nce lossis txo qhov ntev ntawm DNA tawg rau 2-4 feeb raws li lawv tus kheej xav tau txhawm rau ua kom zoo dua cov DNA tawg nrog qhov xav tau loj.

A-2

a) Fragmentation time is not optimized If the fragmented DNA is too small or too big, please refer to the Guidelines for Fragmentation Time Selection provided in the instruction to determine the reaction time, and use this time point as a control, ntxiv rau teeb tsa cov txheej txheem tshuaj tiv thaiv kom ncua lossis luv 3 feeb kom hloov kho kom raug dua ntawm lub sijhawm tawg.

A-3

Qhov txawv me me faib ntawm DNA tom qab kev kho ua kom tawg

a) Txoj hauv kev yaj tsis raug ntawm kev faib cov tshuaj reagent, lossis cov tshuaj reagent tsis sib xyaw tom qab yaj tag. Thaw 5 × Fragmentation Enzyme Mix reagent ntawm dej khov. Thaum thawed, sib tov cov tshuaj reagent los ntawm maj mam ua rau hauv qab ntawm lub raj. Tsis txhob vortex lub reagent!

b) Cov qauv DNA suav nrog EDTA lossis lwm yam kuab paug Depletion ntsev ions thiab chelating cov neeg sawv cev hauv qib DNA kev ua kom huv yog qhov tseem ceeb tshwj xeeb rau kev sim ua tiav. Yog tias DNA tau yaj hauv 1 × TE, siv txoj hauv kev uas tau qhia hauv cov lus qhia kom ua kom tawg. Yog tias EDTA cov ntsiab lus hauv kev daws teeb meem tsis meej, nws raug nquahu kom ntxuav cov DNA thiab yaj nws hauv cov dej deionized rau cov tshuaj tiv thaiv tom ntej.

c) Thawj qhov tsis raug ntawm DNA qhov loj me ntawm DNA tawg ua feem sib cuam tshuam nrog cov nyiaj ntawm DNA nkag. Ua ntej kho qhov kev faib ua feem, ntsuas kom raug ntawm DNA siv Qubit, Picogreen thiab lwm txoj hauv kev yog qhov tseem ceeb los txiav txim siab pes tsawg ntawm DNA hauv cov tshuaj tiv thaiv kab mob.

 d) Kev npaj ntawm cov tshuaj tiv thaiv kab mob tsis ua raws li kev qhia Kev npaj ua cov txheej txheem ua kom tawg yuav tsum tau ua ntawm cov dej khov nruj me ntsis raws li cov lus qhia. Yuav kom ntseeg tau tias muaj txiaj ntsig zoo tshaj plaws, txhua yam tshuaj tiv thaiv yuav tsum tau muab tso rau hauv dej khov thiab kev npaj cov tshuaj tiv thaiv kab mob yuav tsum tau ua tom qab ua kom txias. Tom qab kev npaj ua tiav, thov flick lossis pipet kom sib xyaw kom huv. Tsis txhob vortex!

1. Txoj kev sib xyaw tsis raug (vortex, kev ua phem qias neeg, thiab lwm yam) yuav ua rau muaj kev faib khoom tsis txaus ntseeg ntawm cov tsev qiv ntawv tawg (raws li qhia hauv daim duab hauv qab no), yog li cuam tshuam rau lub tsev qiv ntawv zoo. Yog li ntawd, thaum npaj Fragmentation Mix cov tshuaj tiv thaiv, thov maj mam muab cov raj tso rau saum thiab nqes los sib xyaw, lossis siv tus ntiv tes ntiv tes los nplawm thiab sib tov sib npaug. Ceev faj tsis txhob sib xyaw nrog vortex.

2. High purity DNA yuav tsum tau siv rau kev tsim tsev qiv ntawv

■ Zoo DNA ncaj ncees: Cov electrophoresis band muaj ntau dua 30 kb, yam tsis muaj tus tis

D OD260/230:> 1.5

D OD260/280: 1.7-1.9

3. DNA cov nyiaj yuav tsum yog qhov tseeb Nws tau hais qhia kom siv Qubit thiab PicoGreen txoj hauv kev los ntsuas DNA, ntau dua li Nanodrop.

4. Cov ntsiab lus ntawm EDTA hauv DNA daws yuav tsum txiav txim siab EDTA muaj kev cuam tshuam loj rau cov tshuaj tiv thaiv tawg. Yog tias cov ntsiab lus ntawm EDTA siab dua, kev ua kom huv DNA yuav tsum tau ua ua ntej qhov kev xeem tom ntej.

5. Kev faib cov tshuaj tiv thaiv tawg yuav tsum tau npaj rau ntawm cov dej khov Cov txheej txheem ua kom tawg yog ua rau cov tshuaj tiv thaiv kub thiab sijhawm (tshwj xeeb yog tom qab ntxiv cov tshuaj ntxiv). Txhawm rau kom ntseeg tau qhov tseeb ntawm lub sijhawm tshuaj tiv thaiv, thov npaj cov tshuaj tiv thaiv kab mob ntawm cov dej khov.

6. Lub sij hawm cov tshuaj tiv thaiv tawg yuav tsum muaj tseeb Lub sij hawm cov tshuaj tiv thaiv ntawm kev faib ua pawg yuav cuam tshuam ncaj qha rau qhov loj ntawm cov khoom tawg, yog li cuam tshuam qhov faib loj ntawm DNA tawg hauv lub tsev qiv ntawv.

1. Hom kev piv txwv twg haum rau cov khoom siv no?

Cov qauv piv txwv ntawm cov khoom siv no tuaj yeem yog RNA tag nrho lossis ua kom huv mRNA nrog kev ntseeg RNA zoo. Yog tias tag nrho RNA tau siv los tsim lub tsev qiv ntawv, nws raug nquahu kom siv rRNA cov khoom ua kom ploj (Cat#4992363/4992364/4992391) kom tshem rRNA ua ntej.

2. Cov qauv FFPE puas tuaj yeem siv los tsim tsev qiv ntawv nrog cov khoom siv no?

MRNA hauv FFPE cov qauv yuav raug txo qis rau qee qhov, nrog rau kev txheeb ze tsis zoo. Thaum siv cov khoom siv no rau kev tsim tsev qiv ntawv, nws raug nquahu kom ua kom zoo dua lub sijhawm tawg (ua kom luv lub sijhawm tawg lossis tsis ua kom tawg).

3. Siv cov txheej txheem xaiv me me muab rau hauv phau ntawv qhia khoom, dab tsi tuaj yeem ua rau ntu ntu tso tawm kom pom qhov txawv me ntsis?

Kev xaiv qhov loj me yuav tsum tau ua raws nruj raws qib xaiv me me hauv phau ntawv qhia khoom no. Yog tias muaj qhov sib txawv, qhov laj thawj yuav yog vim cov hlaws sib nqus tsis sib npaug rau chav sov lossis tsis sib xyaw ua ke, cov raj xa dej tsis raug lossis cov kua tseem nyob hauv qhov taub. Nws raug nquahu kom siv cov lus qhia nrog kev nqus qis rau kev sim.

4. Xaiv adapters hauv kev tsim tsev qiv ntawv

Cov khoom siv tsim tsev qiv ntawv tsis muaj cov tshuaj reagent, thiab nws tau pom zoo kom siv cov khoom siv no ua ke nrog TIANSeq Ib Leeg-Index Adapter (Illumina) (4992641/4992642/4992378).

5. QC ntawm lub tsev qiv ntawv

Kev tshawb nrhiav ntau lub tsev qiv ntawv: Qubit thiab qPCR tau siv los txiav txim siab qhov hnyav thiab qhov mloog zoo ntawm lub tsev qiv ntawv feem. Kev ua haujlwm yog nruj me ntsis raws li phau ntawv qhia khoom. Kev tsom xam ntawm lub tsev qiv ntawv feem ntau yuav ua tau raws li qhov yuav tsum tau ua ntawm NGS. Tshawb nrhiav ntau lub tsev qiv ntawv faib tawm: Siv Agilent 2100 Bioanalyzer txhawm rau txheeb xyuas lub tsev qiv ntawv faib ntau yam.

6. Xaiv tus naj npawb ntawm kev hloov pauv

Raws li cov lus qhia, tus naj npawb ntawm PCR mus yog 6-12, thiab tus naj npawb ntawm PCR kev xav tau yuav tsum tau xaiv raws li cov qauv nkag. Hauv cov tsev qiv ntawv muaj txiaj ntsig zoo, dhau qhov ua kom nrov nrov feem ntau tshwm sim hauv qib sib txawv, uas tau tshwm sim los ntawm qhov nce siab me ntsis tom qab lub ncov ntawm lub hom phiaj ntau hauv kev tshawb pom ntawm Agilent 2100 Bioanalyzer, lossis pom qhov pom ntawm Qubit qis dua li ntawm qPCR. Me me dhau qhov kev nthuav dav yog ib qho tshwm sim ib txwm muaj, uas tsis cuam tshuam rau lub tsev qiv ntawv ua ntu zus thiab kev txheeb xyuas cov ntaub ntawv tom ntej.

7. Spikes tshwm hauv qhov txheeb xyuas qhov tseeb ntawm Agilent 2100 Bioanalyzer

Cov tsos ntawm spikes hauv Agilent 2100 Bioanalyzer kuaj pom yog vim qhov tsis sib xws ntawm cov qauv, qhov twg yuav muaj ntau qhov tawg ntawm qee qhov me me, thiab qhov no yuav dhau los ua qhov pom tseeb tom qab PCR txhim kho. Hauv qhov no, nws tau hais qhia kom tsis txhob xaiv qhov loj me, piv txwv li teeb tsa kev faib ua feem rau 94 ° C rau 15 feeb ua kom tsis txhob sib kis, qhov uas faib cov khoom faib me me thiab mloog zoo, thiab homogeneity tuaj yeem txhim kho.