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RT-PCR product dilution and electrophoresis - (Nov/09/2006 )

I did conventional RT-PCR and analyze 5 micro L of products by agarose gel electrophoresis stained by EtBr. There was no differences betwen bands ( I was expect it). Then I dilute same products 10 x, but this time I observed different band intensity. Is this accetable to find different mRNA expression? Is it possible to high, but different ammount of cDNA give same band intensity?

Tnx on Your answers and help!

-hjakovac-

QUOTE (hjakovac @ Nov 9 2006, 08:16 AM)
I did conventional RT-PCR and analyze 5 micro L of products by agarose gel electrophoresis stained by EtBr. There was no differences betwen bands ( I was expect it). Then I dilute same products 10 x, but this time I observed different band intensity. Is this accetable to find different mRNA expression? Is it possible to high, but different ammount of cDNA give same band intensity?

Tnx on Your answers and help!


Agarose gels do have a maximum capasity, above which it is difficult to see differences in intenisity. The bands would in that case also look a little "round in the edges" because the entire content of the band cannot move similtaniously for space reasons.

If this is the case in your experiment, then a dilution would make you able to see the difference.
Loading 5 ┬Ál of a PCR reaction do not sound like someting that would normally cause overload in a standatrd gel, though. Of cause, hard to guess since I do not know anything about your reaction.

Everything is possible with PCR.
If, for exapmle primers or nucleotides are limiting factors, different amount of template could give same band intensity.
The really important question is; How do you know you were using different amount the amplified cDNA? Is there anything in the quantification that may give unsecure meassurements?

-Gerd-

I did RT and PCR in the same tube and use 0.2 mM nucleotides and 10 pM each primer for PCR. I did not perform any cDNA meassurments, I only expect different mRNA expression (I get differences on protein level...I know that is no enough reason). Gene is even constitutively pretty high expressed and different band intensity after dilution was occasional observation.

-hjakovac-