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different amount of cDNA in qRT-PCR for target and reference genes


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#1 Soumble

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Posted 11 August 2014 - 11:11 PM

Dear researchers

 

I am working on mRNA level quantification of copper responsive genes in Klebsiella in the absence and presence of the said metal. I synthesize cDNA using 2ug tRNA and the perform qRT-PCR using 2ul of 10x diluted cDNA. I am using 16S RNA as reference gene but its CT values come between 6.5 and 8.0 while those for target genes are about 24 in the absence and 16-17 in the presence of copper. 

Are these CT values lie in reliable range. I have studied somewhere that CT values should be between 15 and 25 . Is this right? Then what should I do?  If I dilute cDNA further then Ct value for target gene in the absence of copper will go beyond Above limit.

 

 Should  I use 100 times diluted cDNA for 16S RNA gene and 10 times diluted cDNA for target gene?

 

If I do so then what about data analysis? Will different dilutions of cDNA (Different amounts of template for reference and target genes) have any affect on data analysis results? I use Pfaffle method for relative quantification. Should I mention some where in calculations about this difference in template amount?

 

 Will it be appropriate or can any one suggest something else? 

 

Regards

 

Dr Soumble



#2 Trof

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Posted 12 August 2014 - 04:18 AM

No, Ct 6 - 8 in by no means realiable. You are right, it should be at least 15.

 

rRNAs are problematic reference genes, for this reason. One possibility (and AFAIK the one that is recommended) is to reduce the primer concentration of 16S primers to make them amplify at higer Ct.

 

Dilution is a problem, since you can be never sure, that you diluted all samples the same. Using the same volume of the same dilution of cDNA should reduce errors to minimum. By diluting it, you may create some.

But if you chose to ignore this, you will just use the Cts as if you didn't diluted anything, the calculation is always relative regarding the whole sample set, so shifting all the Cts of a gene several cycles have no effect on the calculation. The only problem is the dilution itself.

If publishing, you should mention, that reference Cts were using diluted cDNA.

 

ABI assays often use 18S as a reference, I would try to look for their Application notes, someone may have needed to solve this same problem, as this is a problem of all rRNA assays in general.


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#3 Soumble

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Posted 13 August 2014 - 01:40 AM

No, Ct 6 - 8 in by no means realiable. You are right, it should be at least 15.

 

rRNAs are problematic reference genes, for this reason. One possibility (and AFAIK the one that is recommended) is to reduce the primer concentration of 16S primers to make them amplify at higer Ct.

 

Dilution is a problem, since you can be never sure, that you diluted all samples the same. Using the same volume of the same dilution of cDNA should reduce errors to minimum. By diluting it, you may create some.

But if you chose to ignore this, you will just use the Cts as if you didn't diluted anything, the calculation is always relative regarding the whole sample set, so shifting all the Cts of a gene several cycles have no effect on the calculation. The only problem is the dilution itself.

If publishing, you should mention, that reference Cts were using diluted cDNA.

 

ABI assays often use 18S as a reference, I would try to look for their Application notes, someone may have needed to solve this same problem, as this is a problem of all rRNA assays in general.

Thanks a lot for your reply.

 

I have used primers with final Conc. of 1uM each. What do you think how much I should reduce their Conc.



#4 Trof

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Posted 13 August 2014 - 04:44 AM

1uM final concentration is quite high, 0.5uM is commonly used, but can be as low as 0.2uM even for normal PCR reaction. Try going even lower in these concentrations, make several primer dilutions and see what efect in fany, it has on Ct.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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