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Multiplexing miRNA RT for QPCR


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#1 rnarm

rnarm

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Posted 10 February 2010 - 03:24 AM

Hi,
I was wondering whether anyone on the forum has found success in multiplexing a reverse transcription with multiple single Taqman miRNA RT primers (i'm thinking 3 targets in a single recation would be perfect).

The default protocol recommends single primer RT reactions but the primers can be megaplexed because they exist in the megaplex primer pools used for the ABI TLDA screening.

The default protocol for a single RT target reaction is 7 ul RT mix, 3 ul of target specific RT primer and 5ul total RNA (10ng).

I have tried the following; 7ul RT mix, 1 ul of each target RT primer (3 targets in all), and 5 ul total RNA.

The results are quite comparable as I had assumed the RT primers would be in higher than needed abundance, however I would like people opinion on this.

Thanks for your time,

Richard

Edited by rnarm, 10 February 2010 - 03:25 AM.





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