quality of cDNA - (Nov/10/2007 )
I wanted to know that how to check the quality of cDNA synthesized by RT, as I m not getting my PCR products for one of the ten samples tested for house keeping genes. When I repeated the experiment right from cDNA synthesis, some other sample (other than the first) failed to show cDNA and hence the PCR products.
Did you check the quantity of your RNA before the RT. Usually, as long as the RNA is fine and your RT reagent are fresh, you should get good cDNA.
Here are ways you can do it
Do a spec and the ration of OD260/280 should be 2 for pure RNA. between 1.6 and 2 works for me.
Run a RNA gel to view if there is degradation in the sample.
Also, do you have positive control DNA to test the amplification efficiency and specificity of the primers?