Hello Reena, 

You should take 300-500 ng mRNA for cDNA synthesis. Before cDNA preparation you have to checked the 260/280 as well as 260/230 value > 1.9-2. When you are using qPCR reaction always dilute your cDNA 1:10 for good result.

Hope it helps you.

Thanks.

for qPCR it is the purity of cDNA and the concentration that matters. 20-50ng of DNA per reaction works well in most cases. 

good luck

see when cDNA is prepared please use 1 ug of total RNA, mRNA is just 1 to 3 % of total RNA so thats first part secondly the 20 ul cDNA prepared dilut in 60 to 80 ul of nuclease free water before going to be used in PCR

Hello Sandeep,

Why 1ug of mRNA? Why not less than 1ug? 1 ug is too high. 20 ul cDNA prepared dilute in 60 to 80 ul of nuclease free water- means? If dilute 1:10 times of your cDNA samples hope you get better result.

 didn't say 1 ug of mRNA but total RNA saurav 

thank u saurav, sansrity and sandeep... i do quantify it n m getting ratio from 1.8 to 2.0, n secondly i take 1ug of total RNA for cDNA synthesis..As per q-pcr kit only 1 microliter amt is mention but no concentration.