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Trouble shooting RT-PCR


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3 replies to this topic

#1 kwl

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Posted 17 July 2004 - 08:28 AM

Dear members,
I would appreciate any suggestions or ideas from anyone regarding my problem in RT-PCR.
I am trying to look for low level viral transcripts in dorsal root ganglion tissues. I used beta actin as my control in RT-PCR and they were positive. However many times I tried but I still couldn't see any band for the viral gene product :rolleyes: I'm using Oligo dT primers in my RT, but had read some where that gene specific primers might be better for low level transcripts? The primers that I used had successfully amplify DNA for my gene of interest.
Could I say for sure that my RT is working just based on that I've got a band for beta actin?
How I can make sure if there's any viral gene product or they are below the level of detection? And actually I'm doing nested RT-PCR but still nothing coming up.
I've tried to optimise the RT-PCR by doing Mg titration and using different Taq but still to no avail.
I'm willing to write more if you need extra information in order to help me.
Many thanks.

kwl

#2 kawaka

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Posted 17 July 2004 - 11:52 AM

Hi KWL,

I think if there is any transcripts in your RNA sample, you should be able to amplify it in RT-PCR.

I agree that if the copy number of transcripts is low, you can try gene specific primer in your RT reaction or use random hexamer primer.

How many cycles and how much RT reaction do you use for the PCR?
try more cycles and more RT template.

Do you coamplify your target gene and beta-actin?
If yes, amplify them separately.

Are you sure your viral primer works? You said "The primers that I used had successfully amplify DNA for my gene of interest". I don't have much knowledge on viral genes, do primers for DNA works for RNA?

I found a well written article on troubleshooting RT-PCR. It also gives some tips on low-copy RNA (search the article with "low copy")

Specificly, it says "...Treatment of cDNA reactions with RNase H prior to PCR also can improve sensitivity. The presence of RNA could prevent binding of PCR primers. In some extreme cases of longer full-length targets, such as low-copy-target tuberous sclerosis II, it is necessary to treat cDNA reactions with RNase H. But most often it is not necessary, because incubation at 95C during PCR hydrolyzes RNA...

http://www.pharmagen...312/article.pdf

#3 kwl

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Posted 18 July 2004 - 01:38 AM

Dear Kawaka,
Thanks for your reply.

I had tried to do RT from Sensiscript & Omniscript kit (Qiagen) and Superscript III (Invitrogen). They all gave me positive results for beta actin but not viral genes. I did the beta actin control in a separate tube. I'd also tried to increase the cycle no to 50, used more RT template (not exceeding 1/5 of the RT mixture in PCR), but didn't work. In order to make sure my RT-PCR condition works, I tried it in infected cells first and I got bands for my GOI. It just never work in the tissue. It may force me to say that the level is very low but I'm still hoping to see it,....hopefully one day.

I presume the primers that work for DNA will work for the RNA, if the reverse transcription is working ok.

Do you think working with very little RNA (<1 ug) will result in less RT efficiency, and leading to a failure in RT-PCR? But I've used kit that able to make cDNA from less than 50 ng of RNA.

Is there a way to know the copy number of my viral gene? Since beta actin is abundant in the cell, it's no suprise, it's PCR up everytime.


kwl

#4 BioAdventurer

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Posted 18 July 2004 - 03:30 AM

Hi,

I think that the best thing in this situation, in mypoint of view, is to use random hexamer primers, using the Amersham RT kit. I've used it and is excellent.




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