I measured the quality of cDNA using the Nanodrop. I found similar absorbance for No Template Control also. Although the A260/280 ratio is around 1.8, even no template control PCR product is also showing the same ratio? What is the reason behind it? How to know that how much cDNA is formed? I used 1.2% gel electrophoresis but couldn't find the bands much clear (very much faint band). I measured the RNA quality, it was around 1.7-1.8 which was used as template for cDNA synthesis. And, if I go for Gradient PCR using these products using Specific primers, all the bands appear in one single lane. Is it problematic? (picture with gradient PCR at 60 degree Celsius annealing temperature).
Nanodrop measures the random hexamers/primers/dimers in the reactions too. Try to excise your band from the gel and measure then.
Dear Dipendra, what is your purpose for cDNA synthesis, if Real-Time PCR or gene expression, you can equalize RNA concentrations.
The purpose behind is qRT-PCR and gene expression. I used oligo DT for cDNA synthesis but faint bands apppeared. Should I use random hexamer plus oligo DT for better result? How to equalize RNA concentration for cDNA synthesis? I have RNA samples from RNeasy Plant Mini Kit. I used 50 as a factor for RNA measurement? Is it correct? I used RNase free water for elution in the final step. What is the correct procedure to measure RNA quality and quantity?
Dear Dipendra, RNA concentration factor is 40. After calculating RNA concentrations you can equalize RNA concetrations (take same amount of RNA for example 500 ng or 1 ug according to your cDNA synthesis kit) before cDNA synthesis. We prefer random hexamers in cDNA synthesis.
Concentration of RNA sample = 40 x A260 x dilution factor