Exactly. You cannot measure cDNA quantity on a spectrometer. Any nucleic acid but also proteins (enzymes), phenols etc. that you pipetted in for cDNA transcription or are still present in the tube interfere with your absorbance so the value will not be correct.

Thats why you should normalize your cDNA with a reference gene (preferably two to three) to be able to accurately quantify your transcripts.

As is written in the MIQE guidelines its best to normalize at all stages of processing RNA: If possible you should be using the same amount of tissue for extraction, transcribe the same amount of RNA into cDNA and then normalize your data with reference genes.

We use 1 - 2 µg digested RNA for cDNA transcription and dilute it 1:100 for usage in the real-time PCR.

Hopefully this was helpful.

Olá!

I assume all my RNA was converted into cDNA, and then I use 20-40 ng of this hypothetical cDNA mass per reaction. As far as I know, it is not advisable to quantify cDNA by spectrophotometry because RT reaction leftovers may interfere with absorbance readings :)

Exactly. You cannot measure cDNA quantity on a spectrometer. Any nucleic acid but also proteins (enzymes), phenols etc. that you pipetted in for cDNA transcription or are still present in the tube interfere with your absorbance so the value will not be correct.

Thats why you should normalize your cDNA with a reference gene (preferably two to three) to be able to accurately quantify your transcripts.

As is written in the MIQE guidelines its best to normalize at all stages of processing RNA: If possible you should be using the same amount of tissue for extraction, transcribe the same amount of RNA into cDNA and then normalize your data with reference genes.

We use 1 - 2 µg digested RNA for cDNA transcription and dilute it 1:100 for usage in the real-time PCR.

Hopefully this was helpful.

Doing a full dilution profile of a representative sample mixture (serial 1:2) for 25 points is the first thing to do to visualize where the valid dynamic dilution range is for each study/(per) sample prep method used/(per) target studied.

One is absolutely grasping in the dark if this is not done first every time, for each new experimental situation.

Also, guessing at or stating what dilution of cDNA (pre-qPCR) to use should be avoided; I have experienced situations wherein a 1:1200 dilution of cDNA before qPCR was absolutley required before the log-linear valid dynamic dilution range was reached.

There is no blanket reality for all possible qPCR situations. (Unfortunately)

What constitutes a "representative sample mixture"? And what primer pair would you use for that if you study - lets say - 15 to 20 genes?

I run my standard curves with a sample mixture, using all the genes I study running parallel in the same pcr block. Its the only method I find accurate so far to assess amplification efficiency because of run-to-run variation. One point of my standard curve represents the "real" dilution of the samples in the pcr tube.

I am thinking though that using a sample mixture can mask the effect of inhibitors that might be present in one sample but not in others.

So doing it right would mean to test each sample with a standard curve to assess for pcr inhibition in my opinion, even if that is very time consuming and costy.

I think stating a cDNA dilution in combination with the amount of RNA used to transcribe is okay because it gives the beginner a starting point of what might be possible. Of course it strongly depends on the number of copies of your gene and you might experience that people use something from 1:5 till 1:2000 as their dilution. I agree its irrelevant if you don't know the tissue/species-type, original amount of RNA used for transcription and the kit it was performed with.

Well, I used to work with 10, 40 or 80ng of cDNA. I agree that the spectrofotometer is not the correct way of quantification of cDNA because of the interference of transcription reaction's components. Also, I've never tried to use a mock reaction as blank instead of DEPC water. I do not think that is possible to suppose the cDNA amount based on the RNA input because, in my case, we use total RNA for reaction, not only mRNA. I like the suggestions of normalize the cDNA and work with a serial dilution! I'll try it. Thank you all, guys!!

I've tried both. First of all it depends on you sample. If you now that your genes are not high expressed, is better to make a dilution of the cDNA product and use 1/50-1/100. In other cases, I use 10ng/25ul reaction, both for SYBR Green as well as for TaqMan. You should try to make a standard curve for two samples, one which has higher gene expression and the other with the lowest gene expression. Use more than 7 serial dilutions, and you would have an estimation of the quantity you should use.

And when you make absolute quantification, How to normalize the cDNA? 

Dear Kartar Singh!

 First u can construct cDNA using 2microgram of RNA with Life tech cDNA Kit (high fidelity), and dilute 1microL in to 20 microL DEPC water, then u can take 1microL of diluted cDNA for 3x10=30 reaction mix of qRT and make it easy, u will be get Ct values is19-28, melt curve will be 15-27. u can get very good amplification, melt curve and Ct values with out multiple amplifications peaks... Yes, u can dilute the cDNA and nano gram of cDNA is sufficient to study the expression of genes. if u used diluted cDNA it will be better melting curve and avoid the multiple templates  etc.. nothing will possible to comes errors during the dilution..

Hello Luz maria, You can try to isolate the RNA in same amount of cells or tissue and quantified the every samples in triplicate then calculated  for same concentration of RNA to cDNA construction. if can u  fallow these all, RNA quantification will be more accurate and cant normalize the cDNA for qRT-PCR. Further u can normalize the target  genes with house keeping gene..

Dear:-

it is necessary to dilute the cDNA sample, since for most genes the cDNA is too concentrated for qPCR. Moreover, to calibrate a qPCR reaction for a new gene use a set of about 4 dilutions (for example 4 tubes of 3-fold or 4-fold dilutions) in order to determine the linear range of your reaction ( a range of concentrations in which each cycle doubles the DNA amount). Good luck.