8 replies to this topic

[AnnaSh]

Hi! I have a doubt about the cDNA quantification prior to real time pcr reactions. I'm going to explain. I collected six RNA samples from subsequent fractions of rat small intestine to quantify transcript levels related to a specific protein. 0.5 ug of each RNA were retrotranscribed in parallel, but I decided to control if the efficiencies of RT reactions were the same for each sample. So I tried to quantify cDNAs using the spectrophotometric method (Abs 260 nm), to limit errors when conducting Real Time reactions. The question is: I necessarily have to treat cDNA samples with RNase before quantifying or I can avoid this step? Thanks in advance for your replies.  

[tea-test]

I wouldn't do it. Withiin your RT reaction you still have the RT enzyme (protein), the dNTPs and the primers left which will all give a signal.

[littleaxt]

Tea-test is right, have a look under the pinned topic "Normalization for RNA or cDNA during two step RT-PCR?"

AnnaSh, on Apr 25 2009, 07:36 PM, said:

Hi! I have a doubt about the cDNA quantification prior to real time pcr reactions. I'm going to explain. I collected six RNA samples from subsequent fractions of rat small intestine to quantify transcript levels related to a specific protein. 0.5 ug of each RNA were retrotranscribed in parallel, but I decided to control if the efficiencies of RT reactions were the same for each sample. So I tried to quantify cDNAs using the spectrophotometric method (Abs 260 nm), to limit errors when conducting Real Time reactions. The question is: I necessarily have to treat cDNA samples with RNase before quantifying or I can avoid this step? Thanks in advance for your replies.  

[AnnaSh]

Thanks a lot for your helpful indications! I found in "Normalization..." very interesting suggestions (thanks littleaxt!!!!   ), but I was thinking about the possibility to obtain purified cDNA samples by a simpler method. I was thinking about cleaving the RNA template by RNase incubation and then remove nucleotides and remaining random examers from RT mix through spin columns...Could it be acceptable before quantifying spectrophotometrically? I hope so. I'm agree Tea-test is right, but, based on my experience, even if you use an housekeeping gene to normalize, you can obtain unreliable results: for example, gadph expression is not so "stable" under different stimuli. So I prefer using exactly the same amounts of cDNA for each sample.

Be patient, guys!!!!!!!!!!!!

[gfischer]

You could try using something like Invitrogen's PicoGreen DNA QuantIt kit.  It uses a dye that intercolates into the double helix of DNA and therefore shouldnt be affected by RNA or dNTPs.  Note: you will need a fluorescence plate reader with the right filters.

[littleaxt]

Sorry, but isn't cDNA single stranded? So there won't be a double helix.

And you should also keep in mind, that all these processing steps you want to make prior your qPCR might affect your cDNA. You might loose some cDNA and therefore your result wouldn't be much better. Why not finding a better reference gene if gapdh is not good enough?

gfischer, on Apr 27 2009, 09:59 PM, said:

You could try using something like Invitrogen's PicoGreen DNA QuantIt kit.  It uses a dye that intercolates into the double helix of DNA and therefore shouldnt be affected by RNA or dNTPs.  Note: you will need a fluorescence plate reader with the right filters.

[AnnaSh]

I know that after retrotranscription, you have a hybrid DNA:RNA, that is usually referred as cDNA. Only if you use RNase H, you can degrade the RNA template and obtain a single strand molecule. I surely will try with another housekeeping gene (actin or tubulin), but, based on my experience with semi-quantitative pcr, I think that an accurate quantification of cDNA, although reducing the sample availability, allows more precise results. Furthermore, you can conduct real-time pcr analysis also with a few ng cDNA (20-50).

[gfischer]

littleaxt, on Apr 28 2009, 01:43 AM, said:

Sorry, but isn't cDNA single stranded? So there won't be a double helix.

Sorry, it was a long day.  

[littleaxt]

No, I'm not a molecular biologist, I come from an ecological background therefore I'm never too sure with these things. So I just asked

gfischer, on Apr 28 2009, 04:10 PM, said:

Sorry, it was a long day.