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Reverse Transcription 3-5

Reverse Transcription for the 18S Amplicon

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 from the TaqMan Reverse Transcription

Reagents prime total RNA samples for reverse transcription using

MultiScribe Reverse Transcriptase.

Recommended

Template

Use total RNA samples to generate cDNA for the 18S amplicon.

The following table lists the known template incompatibilities:

Template Quality

The quality of your results is directly related to the purity of your RNA

template. Therefore, use only well-purified samples for 18S. Because

ribonuclease and genomic DNA contamination are common problems

in gene expression studies, purify your samples accordingly to ensure

the best results.

Template Quantity

If possible, use spectrophotometric analysis to determine the

concentrations of purified total RNA samples before reverse

transcription. The table below lists the recommended range of initial

template quantities for the RT step.

Template Explanation

Poly A

The 18S rRNA endogenous control assay cannot accurately

evaluate cDNA generated from poly A

RNA samples

because most of the rRNA has been removed from them.

Non-human Except for 18S rRNA, all assays are human-specific.

Initial Template

Quantity of Total RNA

(per 100-µL RT reaction)

Human Total RNA 60 ng to 2 µg

1 2 ... 24 25 26 27 28 29 30 31 32 33 34 ... 55 56

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Master GREEN 200 Manual de usuario Pagina 29