Problems with qRT-PCR - (Apr/13/2009 )
Hi everyone, so I have got my cDNA with reverse transcription. The cDNA concentration is around 500ng/uL for my samples. I have diluted them by around 4 times, so its about 125ng/uL. I use 4uL of the cDNA samples for template in RT-PCR (which is 500ng for each reaction). And the outcome is there is no amplification and there is no curve. I would like to know if too high amount of template will retard the reaction. My labmate who is experienced with PCR said it won't affect, and he said the more the better. He said it might be the amount of primers and primers affinity that affect the outcome. I am using 3 pico mole of primers (0.06uL of 50uM of each primers). So I would like to ask, what are the possible problems that result in having such a bad result. The melting curve peak is at around 70 something degree. Which is the primer binding region. However, I think that the difference between the ratio of template and primers are too large?? so it affect the reactions? Please give me some insights!! Thanks!!!!
Since You did not mention your reaction volume, there is no way to judge the appropriateness of your reaction components. Did you get amplification from your control primer for housekeeping gene? Have you tested your primers and your cDNA by regular RT-PCR? I think there is a wide working range for template and primers, you should first make sure the primers work, and your RT reaction is successful.
Oh, I am sorry that i forgot to mention about the reaction volume. each reaction is in 20uL, and I have a control using RPL19 primers. Both the housekeeping gene and the gene i want to study doesnt work. How to check if my RT works or not??
in real time PCR less is often more. try 5 ng with your HKG and report your result here. I have already seen strong inhibitory effects with much less than 500ng. do you know that these RPL19 primers are working?