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Help with multiplexing!


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#1 ForEpi

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Posted 01 February 2010 - 11:24 AM

Hi,

I'm looking for a method that can detect between 3 to 9 miRNAs in a single reaction. I thought of a Taqman (or similar chemistry) assay but can't find a reference for it.

Can you help?

Thanks
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#2 rnarm

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Posted 23 February 2010 - 07:13 AM

Hi,

I wrote a post asking the same thing a couple of weeks ago and would too like some input on the matter. What I will say is that the single miRNA QPCR assays are the same primers used in the multiplex primer pools they sell (I asked ABI) so they can be multiplexed.

As a starting point I think I'm am happy enough to multiplex 3 targets in a single RT reaction. The default protocol is 7 ul RT master mix (containing 4.16 ul H2O), 5 ul total RNA and 3 ul specific primer.

What I have tried with success is using 2 ul of 3 target miRNAs and taking the added volume from the water in the RT mastermix.
So the reaction volume becomes 4 ul RT (containing 1.16 ul H20), 5 ul Total RNA and 2 ul of each target primer (6ul total).

When I compare multiplexed with single reaction RT samples they are comparable. Hopefully you might find this works for you too and can reply with your findings.


Regards,

Richard

Edited by rnarm, 23 February 2010 - 07:15 AM.


#3 Fizban

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Posted 24 February 2010 - 07:49 AM

Hi,

I'm looking for a method that can detect between 3 to 9 miRNAs in a single reaction. I thought of a Taqman (or similar chemistry) assay but can't find a reference for it.

Can you help?

Thanks


Multiplexing with taqman probes from ABI is not a good idea in my opinion (mind that i've been using them for nearly 4 years now). first reason is that you have to validate each combination with single assays each time, that's time and money consuming. Second reason is that there are other companies that do not use specific primers for RT (Exiqon (finally!) and Qiagen are 2 examples but there are others.
If you have to do a lot of multiplexing shift to sybr assays. i have no experience with them but i i have every intention to shift to exiqon. still have to test their assays but should be good.
Let us know if u try anything working good
Fiz

#4 rnarm

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Posted 26 February 2010 - 06:16 AM

Hi,

I'm looking for a method that can detect between 3 to 9 miRNAs in a single reaction. I thought of a Taqman (or similar chemistry) assay but can't find a reference for it.

Can you help?

Thanks


Multiplexing with taqman probes from ABI is not a good idea in my opinion (mind that i've been using them for nearly 4 years now). first reason is that you have to validate each combination with single assays each time, that's time and money consuming. Second reason is that there are other companies that do not use specific primers for RT (Exiqon (finally!) and Qiagen are 2 examples but there are others.
If you have to do a lot of multiplexing shift to sybr assays. I have no experience with them but i i have every intention to shift to exiqon. still have to test their assays but should be good.
Let us know if u try anything working good
Fiz




Hi,
Thanks for your input. Can I ask whether you have seen variation in using A taqman multiplex RT? It's just, using the described method above in a few examples I have not seen variation using 2 ul each primer and reducing the water volume in the reaction to compensate.

I understand that he might as well go the exiqon route, but in terms of megaplexing a taqman RT, I can't help but think the conentrations of primers are in highly in excess in the default protocol and ABI sell custom primer combinations anyway.

Thanks,
Richard




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