cDNA synthesis - (Mar/04/2011 )
I'm going to do my RT-PCR. but before that I have to synthesis my cDNA. The problem is that I have a low concentration of RNA. For example, I have a concentration of 44.3ng/ul in 35 ul of sample (S1, so total yield of RNA is 1.5ug and 31.5ng/ul in 35ul of sample (S2) so, total yield of RNA is 1.1ug. Both S1 and S2 are from the same samples. THen, the protocol from the bioline (from cDNA synthesis kit) used 1ug of RNA in a total volume of 10ul of DEPC water. Can someone help me on how to synthesis my cDNA.. Cna I mix S1 with S2 so that I can have a more high yiled of RNA??
Your kit states you can use maximum 10 ul of RNA, so if you can't concentrate it, you have to use 10ul (440 and 315 nanograms respectively) instead of 1 ug. It could be sufficient, depends on the abundance of your target mRNA.
By mixing both your samples you only get more ul of the same low-concentrated RNA, so I don't see any point in doing that.
I have synthesized my cDNA, did the PCR reaction and run the gel, unfortunately, no band appeared accept for my housekeeping gene (ubiquitin). I used 1ug and 2ug of RNA to synthesize my cDNA. I dont know what is wrong. I have also tried using DNA to check whether my primer is working or not. The good news is that, I got bands for my PCR product using DNA sample. And then I have repeated again today using the same sample of my DNA and I ran the gel together with my PCR product from cDNA. suprisingly, no bands appeared for both samples (PCR products from DNA and cDNA). Does anyone know why this was happen???
So you tested your cDNA on a housekeeping gene and it was OK? Was the band thick of faint? If there was a lots of product, that would mean your cDNA is good.
Now testing primers for cDNA on DNA is usually not a good idea. DNA has introns, cDNA not. Are your primers within the same exon (or spanned with only a very small intron)? If not, DNA should not amplify at all.
Is your sample supposed to have high or low amount of your target gene mRNA? Can you get a sample, that surely has enough of your transcript and test your primers on it?
There are lots of reasons, why can PCR suddenly fail, try it again if you have enough of cDNA and be sure you don't omit anything in your reaction, that your primers and mixes aren't old or stored wrong (or just prepare new primers).