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Real Time PCR issues


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#1 jay0306

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Posted 22 July 2009 - 03:50 PM

Hi All,

I am new to this BioForum!I have been trying to do some Real time PCR.I isolate total RNA from normal and tumor tissues using the Trizol(Invitrogen)protocol.I checked my conc. of RNA which gives me 4000ng/100ul in a ND1000 Nanodrop.I use AMV reverse transcriptase using OligoDT primers and later dilute my cDNA 1 to 5 times and run the Real time pCR.I do get expected PCR product of 80-90basepairs.Now my problem is I get a Ct value difference of 7-8 cts(Normal around 27 and tumor around 18-20).The same happens for my normalizing genes as well.Hence I am not able to find a good normalizing gene.As we were suspecting contamination problem I went ahead and did a native RNA gel electrophoresis and as I used 40ng I got a smear and not well organized bands.

My question is as I am getting a PCR product of expected size do I need to worry about the smear on the RNA gel?Or is it b'cos I used such a high concentration of RNA I am getting a smear?I am also planning to do a Bio-analyzer check to get a RIN number.Any suggestions will be really helpful as I want to sort out this issue.
Thanks

#2 cellcounter

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Posted 22 July 2009 - 07:33 PM

Hi All,

I am new to this BioForum!I have been trying to do some Real time PCR.I isolate total RNA from normal and tumor tissues using the Trizol(Invitrogen)protocol.I checked my conc. of RNA which gives me 4000ng/100ul in a ND1000 Nanodrop.I use AMV reverse transcriptase using OligoDT primers and later dilute my cDNA 1 to 5 times and run the Real time pCR.I do get expected PCR product of 80-90basepairs.Now my problem is I get a Ct value difference of 7-8 cts(Normal around 27 and tumor around 18-20).The same happens for my normalizing genes as well.Hence I am not able to find a good normalizing gene.As we were suspecting contamination problem I went ahead and did a native RNA gel electrophoresis and as I used 40ng I got a smear and not well organized bands.

My question is as I am getting a PCR product of expected size do I need to worry about the smear on the RNA gel?Or is it b'cos I used such a high concentration of RNA I am getting a smear?I am also planning to do a Bio-analyzer check to get a RIN number.Any suggestions will be really helpful as I want to sort out this issue.
Thanks


1. Use denaturing agarose gel (formaldehyde) or get a bioanalyzer report for integrity of RNA.

2. In my experience, integrity of RNA is not that important if you are using random primers for First strand synthesis, but with OligodT primers, it is of paramount importance (for obvious reasons). So, best thing would be isolate great RNA, but if you can not do that, use random primers for FSS.

3. Try out many different housekeeping genes. If somebody has done a study on best housekeeping gene for your cell line/organism, use that study to your advantage. Otherwise, do a study of your own.

#3 jay0306

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Posted 23 July 2009 - 09:30 AM

Thanks for the reply.I have no issues with OligoDt primers.I do get my cDNA and then I am able to amplify my pcr product.I have also the PCR product on the gel.So my question is should I need to care about my RNA integrity if I do get a PCR product?Also has anybody worked on Normal and Tumor tissues-any suggestions on which genes will be good as normalizing genes?
Thanks

#4 jay0306

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Posted 23 July 2009 - 09:31 AM

Normal and Tumor colonic tissues?Any Normalizing gene suggestions?

#5 cellcounter

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Posted 23 July 2009 - 11:02 AM

Thanks for the reply.I have no issues with OligoDt primers.I do get my cDNA and then I am able to amplify my pcr product.I have also the PCR product on the gel.So my question is should I need to care about my RNA integrity if I do get a PCR product?Also has anybody worked on Normal and Tumor tissues-any suggestions on which genes will be good as normalizing genes?
Thanks


OligodT primers will give you some product, not all product, if your RNA has degraded a bit.

Seeing a PCR product on gel just proves that your primers are right, and that you have gene expression. It does not tell you whether you are amplifying from all gene-specific transcripts present in your RNA.

You are interested in quantifying the mRNA, so you can not take chances with degraded RNA and OligodT. If you are not planning to get very good RNA, do random priming FSS.

#6 jay0306

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Posted 24 July 2009 - 11:35 AM

Thanks.I will try oligoDt and FSS.

#7 jay0306

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Posted 24 July 2009 - 11:37 AM

Sorry I meant random primers and FSS




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