Hello Daniel

• with the concentration of RNA provided 0.5ul should provide you with robust and consistent cDNA synthesis
• Generally speaking with new iterations of reverse transcriptase 200ng-500ng is sufficient for efficient cDNA synthesis; hence my figure for your volume
• The important thing is that what ever volume in this range you elect to use keep the same for all biological replicates in order to make meaningful gene expression comparisons
• To answer your actual question ‘ how do you quantitate your actual cDNA‘ you can do this in one of two ways

• I might add one more tangential point: if you are proposing to measure cDNA itself for real time gene expression studies then it offers no real advantage over simply putting in a fixed amount of total RNA into the RT reaction and then using the same volume of resulting cDNA in qPCR. I have done qPCR as described but also by column purifying the cDNA and then measuring directly by Nanodrop and using a fixed amount of cDNA in real time gene expression studies. I never found the latter any more reliable
• If however your quest to measure cDNA is unrelated to my supposition then to rev iterate fluorometry + spec with impurified cDNA or Nanodrop measurement at 260nM but with column purified cDNA will both work ( I have successfully done both)

Hello Daniel

• with the concentration of RNA provided 0.5ul should provide you with robust and consistent cDNA synthesis
• Generally speaking with new iterations of reverse transcriptase 200ng-500ng is sufficient for efficient cDNA synthesis; hence my figure for your volume
• The important thing is that what ever volume in this range you elect to use keep the same for all biological replicates in order to make meaningful gene expression comparisons
• To answer your actual question ‘ how do you quantitate your actual cDNA‘ you can do this in one of two ways

• I might add one more tangential point: if you are proposing to measure cDNA itself for real time gene expression studies then it offers no real advantage over simply putting in a fixed amount of total RNA into the RT reaction and then using the same volume of resulting cDNA in qPCR. I have done qPCR as described but also by column purifying the cDNA and then measuring directly by Nanodrop and using a fixed amount of cDNA in real time gene expression studies. I never found the latter any more reliable
• If however your quest to measure cDNA is unrelated to my supposition then to rev iterate fluorometry + spec with impurified cDNA or Nanodrop measurement at 260nM but with column purified cDNA will both work ( I have successfully done both)

If you use a DNase during your RNA extraction, then your total sample should only have RNA in it. Then after RT-PCR you can measure your DNA concentration using Qubit Fluorometer kit to detect DNA and measure concentration. Not an equation, but it works.

Thank you all for your time.

Laurence, I went with the 0.5uL and the PCRs are better than I expected. I still have a bit of optimising to do but this was a great help.

Thanks for the link, Mikael. Angelica, we have a Qubit in house but we don't have the reagents for cDNA analysis.

I'll file this one under quick and dirty but it worked fine.

Thanks again,

Daniel

Dear Daniel, as Sir Laurence already mentioned several approaches to achieve the goal you are interested in. However, I would like to add some practical comments or suggestions to simplify more.

• You must have any fluorometric detection system like Qubit in your lab.
• Select the best DNAse treatment system, which targets only ds strands to assure the removal of gDNA only and not any RNA. For this, you can look for ezDNAse system for reference details (Thermofisher).
• After gDNA removal, check the contaminants and purity levels of RNA through nanodrop and if they are fine, then quantify the RNA though Qubit by Qubit RNA detection kit. And standardize your RNA input as per the maximum enzyme capacity you are using for the reverse transcription setup, (as mentioned for your cDNA kit and is based on enzyme type). Like 500 ng or 1 ug or else, as it also depends on your RNA sample quantity and concentrations mainly. I usually use maximum if I have good RNA sample, like 2 ug of RNA. With SS IV enzymes it can go as high as 5 ug of RNA even.
• If your cDNA kit generates ds cDNA (like for cloning purposes) measure it through Qubit ds DNA detection kit. And, if the cDNA is ss like for qPCR application, so quantify by using Qubit ss DNA detection kits.
• You must have any control cDNA (which are easily available) to check the calibration status of your Qubit device before proceeding any quantification of your cDNA.
• For your reverse transcription, also setup some reactions as negative control, means without RNA template, just add water, buffer, primers / dNTPs and enzyme and also check them through Qubit ds and ss DNA kits along with your normal cDNA samples. Which, will further assure the absence of gDNA in your samples and since it is a negative control, so that readings (only if you get any) can be used to subtract from the cDNA readings you get from your samples through the Qubit to achieve some authentic conclusion about the final readings of your cDNA samples.

Always keep in mind, that cDNA synthesis by any reverse transcriptase is not the exact conversion from RNA to cDNA, rather it is a denovo synthesis of cDNA using RNA as template only. So there is no fix rule that xyz amount of RNA will give you xyz cDNA.

Regards....