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Source of RNA for pcr efficiency, when to use ref gene?


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3 replies to this topic

#1 hianghao

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Posted 24 February 2011 - 02:29 AM

I have a few questions regarding qPCR:
To determine the PCR efficiency, should i use RNA from calibrator sample?
I am going to check gene expression in larva after treatment and also adult after treatment. Calibrator sample should be larva & adult respectively. PCR efficiency should be conducted in both larva and adult or just one of them?
I wanna use treatment with acetone (solvent for drugs in treatment) to compare with treatment with drugs. So i use acetone as calibrator sample. However, i fear that acetone also will induce expression, so i want to set not treated sample as another calibrator sample. Must i check the pcr efficiency in both?
Amplification of ref gene need to be conducted once only for each sample or together with every single reaction?

#2 tea-test

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Posted 24 February 2011 - 04:46 AM

Hi,

RNA from calibrator from larva or adult for efficiency estimation is ok. Also to check the PCR efficiency from only one sample (eg. calibrator) and not from every treatment is ok.
Anmplification from the refgene just once should be also ok. (as long as you don't change any experimental parameters like cDNA input amount, reaction volume,...), also it saves much space on the plate and reagents.
tea-test: The artist formerly known as Ned Land

#3 hianghao

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Posted 24 February 2011 - 07:29 PM

Hi,

RNA from calibrator from larva or adult for efficiency estimation is ok. Also to check the PCR efficiency from only one sample (eg. calibrator) and not from every treatment is ok.
Anmplification from the refgene just once should be also ok. (as long as you don't change any experimental parameters like cDNA input amount, reaction volume,...), also it saves much space on the plate and reagents.


So the ref gene is only amplified once (triplicate) for each RNA source, or each treatment? Because i have 2 RNA extraction samples from each treatment.

#4 tea-test

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Posted 01 March 2011 - 08:53 AM

for every cDNA you have you need the ref genes, but just once. if you do new cDNA from your RNA you have to measure again the ref genes.
tea-test: The artist formerly known as Ned Land




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