I had a 700ng/ul total RNA and got 1432ng/ul of cDNA (measured using Nanodrop-Thermo Fischer). Reverse transcription was done using the Improm II reverse transcription kit (Promega). Is it advisable to repeat the reverse transcription to get >1500ng/ul by optimising MgCl2 concentration or temperature?
Hey,
Instead of concentration of cDNA, do you actually mean quantity? I typically used 3 ng of RNA equivalent to measure miRNA with the miScript System and I know directly from Qiagen R&D that they internally routinely used 1 ng. For mRNA, I typically used 10 ng of RNA equivalent. Keep in mind that the reverse transcription step not 100% efficient so you may not have as much cDNA as you think. Also, many reverse transcriptase degrade the template RNA as they convert it into cDNA. This suggests that you cannot get more cDNA than you initially have of RNA. I am not familiar with Improm II however. I also want to point out that the quantification of your RNA was carried out by a Nanodrop which relies on absorbance and the A260 absorbance depends on pH. Did you use the same solvent for your RNA and cDNA absorbance readings? If not, this could explain why you think you have more cDNA than RNA?
Hope this helps.
Jean-Charles
Thanks a lot Neel. Lot's of researchers have also discredited the accuracy of absorbance readings of cDNA , given that we have left over dNTPs, and RNA and other reasons. I get the point. The question here is, how do I know whether or not to proceed with PCR with the above results? Can an agarose gel electrohoresis of the cDNA help?
The easiest way to find is by doing the RT-PCR - make sure you include a "no RT" control (which may also help you to understand why you shouldn't bother doing the nanodrop of the cDNA synthesis reaction). The only way to see on a gel whether the cDNA synthesis has worked is to include a radioactive nucleotide and then transfer the gel onto a blot to see a radioactive smear.
Hi Ubanako,
I agree with Matthew. At this point, you do not need to quantify your cDNA reaction product since you indeed cannot distinguish leftover RNA from cDNA. Before deciding to repeat the reverse transcription to increase your total amount of cDNA, you need to decide how many nanograms of initial RNA you want to use per PCR reaction. If you started with 700ng RNA, you might have enough for 70 reactions if you were to use 10ng RNA equivalent. This depends on the expression level of your mRNA of interest within your RNA. I have seen people use as high as 50ng and as low as 7ng for mRNA detection.
Jean-Charles
Bear in mind that the RT step is not necessarily linear: i.e. a RT from 50 ng can result in different apparent mRNA transcript levels compared with a second experiment where you start with 500 ng. Depending on the RT and mastermix you use, 10 ng of RNA reverse transcribed using random primers should be more than enough to detect most transcripts. I would also suggest you use dual pled qPCR assays, since they are twice as informative and the vast majority of PCRs work very well as dualplex assays once optimised. You usually only get problems when you start triple plexing.
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