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multiplexing taqman miRNA reverse transcription


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#1 rnarm

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Posted 10 February 2010 - 03:26 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

#2 Baars01

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Posted 25 February 2010 - 06:15 AM

You have an interesting method. How many ng of RNA do you add to your RT rxn btw? Perhaps adding too much would compromise the primer concentration?

I was trying to discover the same thing. Was thinking of adding another 3 ul of a different primer (endogenous control) and adding less water to the RT mix. Had a chat to an ABI technician who said that it was possible "in theory", but she also mentioned that the other components of the RT master mix could be in too low abundance to facilitate the extra transcription. The technician said she would get back to me about this one.

I have also been trying to find research where they do the same, to no avail.

Edited by Baars01, 25 February 2010 - 06:18 AM.


#3 Baars01

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Posted 26 February 2010 - 07:16 AM

Applied Biosystems said it is worth performing an Reverse transcription and qPCR using the RT primers multiplexed. If experession shows up, this means that the primers dimerise and amplify. If this comes up at a ct of around 38, this is statiscally negligible, but lower than this can influence your results.




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