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need help setting up reverse transcriptase qPCR

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#1 molbio1234

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Posted 11 October 2014 - 08:26 AM

I need help setting up a reverse transcriptase qPCR assay. I am trying to understand why a few of my RNA samples are failing to produce cDNA products after reverse transcriptase reactions. I want to see if reverse transcriptase qPCR will measure any of my RNA material by converting it to cDNA and measuring the amount of cDNA from the assay.

 

From my experiment, I have saved aliquots of my stock RNAs that failed, diluted RNA used as input the reverse transcriptase reactions, after mRNA fragmentation (to produce cDNA products of a specific size), and after final cDNA synthesis. I will measure the final cDNA synthesis products using regular qPCR to measure DNA. I have the same aliquots above for control RNA samples that gave cDNA products.

 

I bought a one step reverse transcriptase qPCR kit and have GAPDH, 28S, and B actin primers. Most people tell me it is the samples but unless I can show that, I can't prove it. I have been able to measure the RNA by RIN score and Qubit and it measures and RIN looks fine. So I don't think it is the samples but something else. I want to see what reverse transcriptase qPCR will show.

 

 

 



#2 bob1

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Posted 12 October 2014 - 01:52 PM

It could be contaminants in the samples coming through the extraction procedure. What are you extracting the RNA from.  Note that RNA and DNA are very similar in terms of how they are measured by things like Qubit, so you could be measuring DNA rather than RNA, but still have a good RIN.



#3 molbio1234

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Posted 13 October 2014 - 07:34 AM

I am using Qiagen RNeasy kit. I also Nanodropped and 260/280 was ~2. I use Qubit RNA kit so it should only be specific to RNA.

 

I need some help setting up reverse transcriptase qPCR though. It is the only assay that is sensitive enough to measure my aliquots at the different steps. Should I use the straight aliquots or dilute to a certain concentration first and place into the assay? What's a good way to set up the assay?



#4 molbio1234

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Posted 13 October 2014 - 02:38 PM

Does anyone have any suggestions on setting up this reverse transcriptase assay? sad.png







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