I treated my RNA sample with DNase and I measured DNA concentration using Nanodrop and I found it was 2.28ng/µl. can I do cDNA reaction to perform qPCR reaction with this small amount of contaminating DNA without going to gel electrophoresis to visualize the bands? what is the maximum amount of contaminating DNA in RNA sample in terms of using Nanodrop to estimate the contamination?
One thing you can try to do is perform a PCR directly on your RNA so you can understand the level of amplification you may get from gDNA. If your gene of interest does not amplify directly from the RNA then you would likely be safe to do RT-qPCR.
Nanodrop measurement can't differentiate DNA from RNA the only difference between the DNA and RNA setting is the factor used to convert OD260 to concentration.
That said your concentration is very low (for total RNA, should be fine if you did polyA selection )so you may not get a good cDNA. Did you nanodrop before the DNAse treatment?
Unless your gene is very abundant it's going to be very hard to get something from qPCR starting with less than 100ng total RNA.
You can estimate the levels of DNA contamination by measuring using Qubit or by running a no-RT reaction in the qPCR (always a good idea).
I agree with Yoav. The device is simply measuring A260 and applying a different conversion factor to the OD to alternate RNA DNA measurements. If you want to check for gDNA contamination you can run a small portion on an agarose gel, gDNA well stay near the well as its huge, while RNA will migrate into the gel.
Thank you all for your answers..Actually I run a gel electrophoresis and I got a blurred smear on the first 2 lines as you can see on the attached picture, the 3rd line is the +ve control and the one next to it is the -ve control..I am using Qubit to measure DNA and RNA concentration and it can diffrenciate between DNA and RNA as it gave me RNA concentration of 180ng/µl and a DNA concentration for the same sample as 2.8ng/µl..So, my question is ''what is the maximum accepted contaminating DNA concentratrion that can be foundin the sample to perform qPCR reaction?''
Thanks
Once you have treated the RNA with DNase. It is very difficult to check for the residual gDNA, but one quality check that could be done is analyzing the RNA on the gel to check for the integrity of the RNA after DNase treatment, and also you can do a PCR with the RNA as a template for a known specific gene. Further there should no amplification with the RNA as a template using the primers for a known specific gene. Qubit check, Gel analysis and absence amplification should be enough to proceed for cDNA conversion. Ideally 500ng of RNA needs to be taken for cDNA conversion. For other applications apart from qPCR, further purification and purity analysis of RNA needs to be carried out to check gDNA contamination.
I looked up a book that says gDNA contamination does not necessarily affect cDNA synthesis. I have included the link. I generally do not worry about that amount of gDNA as long as my RNA yield and quality is excellent.
https://books.google.com/books?id=-s5oRDUuMSIC&pg=PA470&lpg=PA470&dq=how+much+contamination+of+gDNA+is+allowed+in+RNA+in+proceeding+with+a+cDNa+conversion&source=bl&ots=y67AqAwIsv&sig=N0N9B32Gwkkp6Mlf4iuoyavZDto&hl=en&sa=X&ved=0ahUKEwi9z-TepOLbAhXuna0KHQepB1YQ6AEIczAH#v=onepage&q=how%20much%20contamination%20of%20gDNA%20is%20allowed%20in%20RNA%20in%20proceeding%20with%20a%20cDNa%20conversion&f=false
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