I understand that it is not necessary to purify cDNA for real time PCR (as I had some good results previously) but still I want to be really sure.
I will be very grateful if some one share with me a protocol of cDNA clean up. I mean how to purify cDNA from free nucleotide and primer.
Please, see the following link:-
http://www.protocol-online.org/biology-forums/posts/6246.html
Best regard
The quickest/easiest is a Qiaquick PCR purification kit (or equivalent). Macherey-Nagel sells a good kit as well (NucleoSpin Gel and PCR Clean-up). In general it will take you 10 minutes total.
Thanks Dr. Livingstone. I did it by PCR Clean up kit. Then I quantified cDNA by nanodrop and I found that A260/280 is 2 (and not 1.8 that is the value for DNA) in all cases and A260/230 is less than A260/280 (ideally it should be more than A260/280 for DNA and RNA). I assume that the trend for cDNA should be similar to DNA. Am I right? Is it OK to proceed for Real time with this cDNA?
Thnaks!!!
Hello Nidhi,
Why do you want to purify the cDNA? Instead you can purify your RNA and check its 260/280 and 260/230 ratio. BTW I don't think the 260/230 ratio has to be greater than 260/280 for DNA and RNA! Pure RNA should have a 260/280 ratio of 2 but 260/230 ratio of 1.8 is acceptable for NGS applications. And basically the first strand cDNAs are single standed not double standed as a normal DNA so there might be different ratios. Usually when we check our cDNA it shows a 260/280 ratio of 1.8 to 1.9.
Best of luck.
Hi Nidhi,
I did real-time PCR for years without cDNA cleanup and it worked well. However, we lately had problems with variation in technical replicates and in this case the cDNA cleanup really helped a lot.
I can recommend a column-based PCR clean-up kit for cDNA cleanup. We use Genomed's JetQUick PCR Purification kit, but any other should work. Just be sure that you leave out the RNase H step in the cDNA synthesis protocol, as the PCR purification kits are optimised for dsDNA.
Good luck,
Britta
Hello Dr. Banerjee and Dr. Kirbach
I already purified RNA and it is really a good quality RNA with 260/280 of 2. But I was having problems in qPCR carrieed out with the cDNA synthesized from this RNA. I know that cDNA purification is not necessary and evens i never purified it before but as Dr. Kirbach said that he had problems with variation in technical replicates, likewise I am also experiencing some problems, thats why I went for the purification step. Lets see how it goes this time.
Thankyou very much both of you for yours suggestions.
Nidhi
I also checked the functionality of my primers with cDNA and also with genomic DNA and I observed almost similar amplification/expression in both cases. However I was expecting more amplification in cDNA as compared to genomic DNA (due to presence of introns in genomic DNA). Am I correct? I am not able to get a proper justification for it in terms of introns and copy number. Please help me clear this query.
thanks!!!
Hi Nidhi,
do your primers span exon/exon boundaries? If so, you shouldn't get any amplification from genomic DNA, as introns mostly are too large to be easily amplified (or at least, the product should run higher - you can check that on a gel also after real-time PCR). If not, it depends on how much gDNA you put in the PCR reaction and how high your gene is expressed. In my case, I often had more product with gDNA as my genes of interest are expressed at a rather low level. So it might be that you have the same amount of amplicons by chance.
Hope this helps you,
Greetings,
Britta
Dear Dr. Britta,
Thanks for replying!!!
I understand the second part of your explanation, one that says about quantity of gDNA and level of expression of genes of interest. But the first part of explanation is not clear to me. Yes, my primers are from exon region, but I can see amplification in genomic DNA as well and ideally it should amplify the exon region (may be not the entire region) of the gDNA. In my case, I observed amplification in both cDNA and gDNA (as expected), but contrary to my belief, amplification was similar in both cases. However I was expecting more amplification in cDNA. I hope, I was able to explain well my query.
Thanks once again!!!
Regards,
Nidhi
Hi Nidhi,
if you design primers for cDNA you usually take care to let them span two exons, so that amplification from gDNA is hindered by the intron. You can easily check that for your primers in Ensembl or GenBank.
Greetings,
Britta
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