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Comparative semiquantitative RT-PCR


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#1 Nephrite

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Posted 14 September 2011 - 05:18 AM

Hello.

Here is the situation - I collect samples in different time points of my experiment and for each sample I have to evaluate the expression of several genes by means of RT-PCR and measuring the intensity of the bands.

My questions:

1. What is technically more correct - to run all reactions together (incl. the housekeeper) under the same conditions or to run each primer pair separately and choose particular number of cycles?

2. Is it true that the optimal number of cycles for a particular primer pair is the one when a detectable accumulation of amplification product is being started?

3. If the second is true, by optimizing the cycle numbers for each primer pair we try to mimic the qPCR, right?

#2 vitalgene

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Posted 14 September 2011 - 06:12 AM

my opnion on ur question
1) to run primer pair separately, with number of cycles (primer are unique and vairable factor for PCRs, use appropriate anealing)
2) ture, but looking for each set sucha way is a hurculean task so I run at first stand at 30 cycels and depend on intensites decide emphircally the cycle no s
3) in principle yes, but practically they are different techniques and grossly concluding the same.

#3 Nephrite

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Posted 16 September 2011 - 01:04 AM

my opnion on ur question
1) to run primer pair separately, with number of cycles (primer are unique and vairable factor for PCRs, use appropriate anealing)
2) ture, but looking for each set sucha way is a hurculean task so I run at first stand at 30 cycels and depend on intensites decide emphircally the cycle no s
3) in principle yes, but practically they are different techniques and grossly concluding the same.


Thank you! :-)




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