Problems with second use of diluted cDNA in qPCR - (Sep/30/2008 )
Hello All,
Each time that i am using diluted cDNA in my reaction, i am getting a good results.
However, when i use the SAME (from the same tube) diluted cDNA, the results looks like a noise!
I have tried to store the samples in -20C or +4C, the results remain the same. Moreover, during the preparations of the qpcr samples the cDNA has been stored on ice.
The Problem occur consistently.
May be, someone has an idea why does it happening??
Please Help!
Regards,
Anat
What do you dilute with? Could it be contaminated with nucleases?
The dilution of my genes are from 1 to 625 and the house keeping gene (18s) diluted by 1000 to 625000
During the reaction in volume of 20 micro liter this happened only in housekeeping genes, but reactions with 10 micro liter it happened with both of them.
Before the cDNA creation i have treated the RNA with DNase, so it shuoldnt be with nucleases.
The dilution of my genes are from 1 to 625 and the house keeping gene (18s) diluted by 1000 to 625000
During the reaction in volume of 20 micro liter this happened only in housekeeping genes, but reactions with 10 micro liter it happened with both of them.
Before the cDNA creation i have treated the RNA with DNase, so it shuoldnt be with nucleases.
Did you inactivate the Dnase properly before starting your cDNA reaction?
What kind of RT kit are you using?
I didn't use RT kit. I have create cDNA by adding 1micro liter random hexamer and incubate it at 65°C for 5 minutes, when I add buffer for Reverse Transcriptase, RiboLock™ RNase Inhibitor, dNTP Mix, 10 mM each and M-MuLV Reverse Transcriptase and incubate it for 60 minutes at 37°C. Finally, terminated the reaction by heating at 70°C for 10 minutes