( vely low efficiency of t...)HotStarTaq PCR Handbook

vely low efficiency of the reverse transcription reaction must
be considered when calculating the appropriate amount of starting template for subsequent PCR. The volume of the RT reaction transferred should not exceed 10% of the total PCR volume. General guidelines are presented in Table 12 below.

Table 12. General guidelines for performing RT-PCR

RNA purification
and reverse
transcription: QIAGEN offers the RNeasy system for total RNA isolation, Oligotex Kits for messenger RNA isolation, and Omniscript Reverse Transcriptase for reverse transcription.* Follow the detailed protocol in the Omniscript Reverse Transcriptase Handbook. Or, when using an enzyme from another supplier, follow the manufacturers instructions. The following guidelines may be helpful.

Mix the following reagents in a microcentrifuge tube:
4.0 l 5x RT buffer
1.0 l RNase inhibitor (5 units/l)
2.0 l DTT (0.1 M)
1.0 l each dNTP (10 mM)
~1 g RNA
2.5 l primer (0.2 g/l)
reverse transcriptase
Add RNase-free water to a final volume of 20 l.
Incubate following the manufacturers instructions.
Heat the reaction mix to 95C for 5 min to inactivate the
reverse transcriptase. PCR: Prepare a PCR mixtur
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