Hi Saeideh,

I work routinely using qPCR to compare the mRNA levels in neural stem cells and I think I can help you a little bit on the design of your qPCR.

I use 384 well plate and my final volume per well is 6.25 µl:

3.11 µl of Sybr green

0.625 µl of cDNA

1.255 µl of H2O

0.63 µl of Forward primer (working solution at 5µM)

0.63 µl of Reverse primer (working solution at 5µM)

For one reaction, I use 0.625 µl of cDNA obtained from 3.125 ng of RNA by RT-PCR. To make it simple, I use 1µg of RNA and do the RT-PCR in 20µl total volume. I dilute the cDNA 1/10 (200µl total volume), and then I use 0.625µl for each reaction at the qPCR.

If you use 96 well plate, you can use 15µl reaction and change the amount of reagents accordingly.

Also, check that your qPCR program has the right cycles and temperatures for all the different amplifications.

Hope this information helped your research!

Cheers,

Ale

Hi Saeideh,

I work routinely using qPCR to compare the mRNA levels in neural stem cells and I think I can help you a little bit on the design of your qPCR.

I use 384 well plate and my final volume per well is 6.25 µl:

3.11 µl of Sybr green

0.625 µl of cDNA

1.255 µl of H2O

0.63 µl of Forward primer (working solution at 5µM)

0.63 µl of Reverse primer (working solution at 5µM)

For one reaction, I use 0.625 µl of cDNA obtained from 3.125 ng of RNA by RT-PCR. To make it simple, I use 1µg of RNA and do the RT-PCR in 20µl total volume. I dilute the cDNA 1/10 (200µl total volume), and then I use 0.625µl for each reaction at the qPCR.

If you use 96 well plate, you can use 15µl reaction and change the amount of reagents accordingly.

Also, check that your qPCR program has the right cycles and temperatures for all the different amplifications.

Hope this information helped your research!

Cheers,

Ale

Are your primers specific for the target gene? checking the temperature for melting and annealing as Alejandro suggested might help.

2nd thing, the volume of the constituents is of 2nd priority, the concentration matters.

You should not really work with ul, since that tells you nothing about the concentration of your primers. The final primer concentration should be around 300nM for SYBR Green, but can be anything from 150-500. Go to the IDT website (www.idtdna.com), where you will find all the relevant information you require. It is really very very good.

the final primer concentration we use for qPCR is 250 nM for forward and 250nM for reverse and it works good with different templates and different primers. When it comes to cDNA, I use 5 µl template in 25 µl reaction but the dilution I determined each time based on the primer efficiency (standard dilution analysis).

Thanks everybody for your suggestion.

I also have another question.

I have some cDNA samples and I dont know if I should trust the nanodrop concentration for RNA or I should trust the cDNA nanodrop. Because some people say it is better to use the RNA concentration as determining cDNA concentration can be tricky due to the dNTP's in the solution, which will through off measurements and it will also need cleaning the sample. But that would result in lost of material. Instead, they recommend measuring the RNA concentration before the RT step, and then assume a 1 :1 conversion to cDNA. (ie, 1 ug RNA = 1 ug cDNA)

Hi Saeideh,

The old Reverse Transcription (RT) efficiency problem!

Firstly I would caution against any spectrophotometric analysis (Nanodrop or Picodrop etc.) of “cDNA” because in most cases it’s not just cDNA, it’s the reverse transcription (RT) reaction containing cDNA. So as your colleagues described, measurements will be compromised by the reaction constituents such as free dNTPs.

Instead, it’s probably best to run all of the relevant RNA samples of a particular experiment simultaneously, in the same RT thermocycle, preparing samples with a single RT reaction cocktail.

Additionally, if you’re performing absolute quantification, it is essential to standardise your RNA concentrations beforehand using RNase-free water (NOT DEPC-treated water, because DEPC can interfere with PCR). It’s best practice to do this when performing relative quantification too.

After that, we come to the assumption of 100% reverse transcription efficiency. Here is where most labs differ. I was taught to repeat the RT reactions on the full set of samples so I would have 2 RT reactions for each sample. I could then pool each sample’s separate RT reaction into 1 representative RT mix. However, I’ve also worked in labs where only 1 RT reaction was used.

Essentially you’re assuming a 1:1 conversion (or 100% RT reaction efficiency). However because your samples are prepared and run together, any deficiencies in the RT are assumed to affect all samples equally. This allows you to accurately compare mRNA transcript levels accurately across your samples.

As qPCR quantity estimates (even absolute) are ratios, either to a reference gene or a sample with known concentration/copy number, what is most important is to maintain uniformity across all samples. You can confirm success of the RT reaction with a positive control.

Using qPCR to compare samples prepared by different RT reactions introduces confounding factors which are extremely difficult to control for (i.e. RT cocktail variability and differences in thermocycle performance). I would advise against any experimental workflow that requires you to do this unless you have read the attached paper, describing the use of inter-run calibration (IRC).

On a final note, if you wanted to persist with using a nanodrop/picodrop to analyse the RT reaction, I’ve heard researchers describe using RT negative controls as blanks in spectrophotometry with some success. However, I have no personal experience of this.

Good luck!

Paul

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Thanks alot every body for your valuable suggestions.