Amplified Minus RT - (Dec/04/2006 )
I am doing real-time PCR to amplifiy cDNA. Prior to cDNA synthesis i have DNAse treated my RNA to ensure no gDNA contamination.
However today i did an assay and my NTC's where negative, but 2 of the three minus RT controls amplified- giving a Ct value of 40.
The correct Ct for this gene is 29.
One other thing to note is that i am using TaqMan gene assay and an exon spanning assays was not available for my gene of interest so the TaqMan primers and probes are located within an inton.
Do i still have gDNA contamination- even though i did Dnase treatment and only 1/3 minus RT amplified?
Any suggestions on what i can do?
a Ct value of 40 is a very very small amount of genomic DNA. even proprietary RNA extraction kits cannot guarantee 100%removal of DNA contamination. if your gene of interest comes in at 29 i would not be overly worried about an RT- coming in at 40