Difference in RT PCR products when using total RNA or mRNA
Posted 25 May 2002 - 08:14 AM
Since several months, I perform a RTPCR using 12 µg of total RNA as template for RT. For the PCR I used 2µl of the RTproduct. The RTPCR worked very well and gave 5 bands .3 of them were cloned. surprisingly, the 2 others could'nt been cloned.
Since one week, I use 1µg mRNA (isolated with the kitmicrofast track form invitrogen)as template for the RT and I use similar quantities of cDNA as previously but I obtain poor amounts of PCR products and the bands that I couldn't clone before disapear.
Does someone have a solution or explication for this problem? ? Thank you very much
Posted 25 May 2002 - 09:19 AM
Posted 27 May 2002 - 06:53 AM
I've already tried isolating the bands of the gel and reamplify them .But when I do this, I obtain all the five bands as if I've not isolated the bands of interest. The negative controls of RT (PCR with RNA and RTPCR without reverse transcriptase) are negative...so I think that there are'nt genomic contamination!
Posted 30 May 2002 - 10:15 PM
I think the 2 bands you got are due to one oligo amplification only. Did you try to check them separately on the total RNA cDNA?
Posted 16 December 2004 - 07:25 AM
The occurance of DNA in a total RNA isolate usually means you are using too many cells. Try using less tissue, or more extracting media.
Increasing the annealing temperature in the PCR reaction can dramatically reduce any artefacts from the amplification, due to mispriming.