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rnarm's Content

There have been 9 items by rnarm (Search limited from 13-August 19)


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#60524 Help with multiplexing!

Posted by rnarm on 26 February 2010 - 06:16 AM in siRNA, microRNA and RNAi

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



#60022 Help with multiplexing!

Posted by rnarm on 23 February 2010 - 07:13 AM in siRNA, microRNA and RNAi

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



#58357 multiplexing taqman miRNA reverse transcription

Posted by rnarm on 10 February 2010 - 03:26 AM in PCR, RT-PCR and Real-Time PCR

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



#58355 Multiplexing miRNA RT for QPCR

Posted by rnarm on 10 February 2010 - 03:24 AM in siRNA, microRNA and RNAi

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



#55669 MirVana + RNAlater white pellet?

Posted by rnarm on 18 January 2010 - 03:06 AM in siRNA, microRNA and RNAi

That is strange. I'll keep an eye out next time I extract. I would suspect from the 260/280 the pellet contains protein. Perhaps phone ambion (ABI) to see if they could advise with extra wash steps.



#55668 The reliable data of microRNA expression from SYBR-stem loop PCR or ABI Taqman m

Posted by rnarm on 18 January 2010 - 03:00 AM in siRNA, microRNA and RNAi

A word of advice from myself is to discount anything over 35 cycles on the array because speaking with ABI they suggested anything over 35 is not expressed. For my data, I chose a 35 CT as my cuttoff for undetermined, not 40 CT.

I also find the CTs for endo and targets come up later in single assays compared to TLDAs (Im using taqman validation). So a gene which might come up at 30 CT in a TLDA might come up at 35 CT in single assays when using 1 ug total RNA in TLDAs (endo also increases). So select your genes for validation carefully from your TLDAs.



#54896 MirVana + RNAlater white pellet?

Posted by rnarm on 12 January 2010 - 08:32 AM in siRNA, microRNA and RNAi

Hi,

I have no experience with RNA later but have had a white pellet after mirVana extraction. I used to think it was because of a high yield of RNA was recovered but I have had it recently wherby I didnt notice it until after I nanodropped the sample, resuspended and only found a slight change in concentration and decrease in 230 ratio.

I think the wash solutions were getting old as it occurred when solutions were low.



#45618 Question about miRNA-star and Taqman Q-PCR miRNA array validation

Posted by rnarm on 17 November 2009 - 09:54 AM in siRNA, microRNA and RNAi

Hi. I have 2 questions on miRNAs I was hoping someone here might be able to answer. Firstly do miR-star strands target differential mRNAs to the non-star strand. If so, are these targets accounted for in prediction databases e.g. TargetScan pulls miR family targets.

Secondly, I have conducted a few miRNA Q-PCR arrays which I am currently validating. The problem I have is the RT primers for the array are cyclic (40 cycles) but single assays consist of using a single non-cyclic RT reaction. This causes my endogenous control (u6 or RNU48) to come up much later in the cycles compared to the arrays. So my question is, if my endogenous is CT 26 for U6 in the single assays, can anyone here comment on what CT would be moderately expressed miRNA? I ask this because miRNAs I initially selected from my arrays that were coming up at CT 30 (U6= CT19) are coming up too late for single assay validation (CT 37).


Thanks in advance,

Regards,

Richard

:)



#39026 ABI Taqman Low density microRNA Array card

Posted by rnarm on 07 October 2009 - 02:30 AM in siRNA, microRNA and RNAi

Hi LW,

I have been working on the ABI taqman arrays (used over 50). I think it is normal that 190 miRNAs are undetermined in the A array. It is a screening process and I have similar results. Expect even more to be undetermined in array B (possibly getting a flagged plate for well percentage amplification in SDS manager). Also remember, over 35 cycles is classed as not expressed in these arrays!

I am currently having some issues validating my results. This is because the cDNA conversion in the megaplex reaction is over 40 cycles, whereas for a single assay it is only a single conversion. This makes it difficult validating because miRNAs come up later in the cycles for single assays. In the middle of getting help from tech support about this issue.


RNA (that's actually my initials)




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