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tRNA and Real time RT-PCR


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#1 molcyt

molcyt

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Posted 22 March 2005 - 04:37 PM

Hi,
I am trying to determine my gene expression in blood using Real time RT-PCR (sybrgreen). However, realtime PCR produced primer dimer instead of expected product. (Primer is designed in exons on both sides of an intron). I saw a note in a paper about primer-dimers. It was saying that if low espression presents, this can cause primer-dimers. And recommends to use tRNA. Do you have any detailed information about this situation? or do you have any idea how to detect low expression in blood? Thanks a lot.

#2 yongyk

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Posted 23 March 2005 - 01:05 AM

Dear molcyt,
First of all, why your gene has low expression? and why your assay can from primer dimer? (Is your assay not optimized or your primer can form primer dimer?)

I don't know anything about tRNA but can give your a suggestion to get rid of primer dimer.

First when you run melting curve analysis, you should see two peaks. The one with higher Tm is your specific product; the lower Tm is primer dimer.
Let say your melting temperature of the specific product is 85oC and you drimer dimer is 75oC. You can perform your PCR in as following condition
95oC 30s
550C 30s
72oC 30s
80oC 10s*

* set as optical data collection step.

If you collect your optical data at the temperature higher than you primer dimer formation temperature, what ever data collected well be free from primer dimer interference.

so you still can collect a valid data olthough the gene has low expression.

Hope this will work for you. I tried and it work for me.

Best Regards
Yongyk




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