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3-2 Reverse Transcription

Reverse Transcription for All Amplicons Except 18S

Overview

Synthesis of cDNA from total RNA samples is the first step in the

two-step RT-PCR gene expression quantification experiment. In this

step, random hexamers, oligo d(T)

, or sequence specific reverse

primers from the TaqMan Reverse Transcription Reagents

(P/N N808-0234) prime total RNA samples for RT using Multiscribe

Reverse Transcriptase.

Guidelines

Follow the guidelines below to ensure optimal RT performance.

A 100-µL RT reaction efficiently converts a maximum of 2 µg total

RNA to cDNA. Perform multiple RT reactions in multiple wells if you

are using more than 2

µg of total RNA.

Use random hexamers, oligo d(T)

, or sequence specific reverse

primers to reverse transcribe the total RNA samples for gene

expression assays.

The choice of primers for RT is best made after experimentally

evaluating all three priming systems. For short RNA sequences

containing no hairpin loops, any of the three priming systems work

equally well. For longer RNA transcripts or sequences containing

hairpin loops, consider the following guidelines:

Primers Selection Guidelines

Random hexamers Try first for use with long reverse transcripts or

reverse transcripts containing hairpin loops

Use to transcribe all RNA (rRNA, mRNA, and

tRNA)

Sequence-specific

reverse primer

Use to reverse transcribe RNA-containing comple-

mentary sequences only

Oligo d(T)

Use to reverse transcribe only eukaryotic mRNAs

and retroviruses with poly-A tails

Avoid long mRNA transcripts or amplicons greater

than two kilobases upstream

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Master GREEN 200 Manual de usuario Pagina 26