2 replies to this topic

[moljul]

hallo there,

i´m currently doing some transfection stuff and have to quantify vector as well as mRNA afterwards!

when i run my PCR, signal comes at around Ct 23-26 (means that transfection was OK)!
when i perform DNase I pre-treatment before cDNA-Syntehsis and run real time PCR with my RNA as template, product signals come at 33-35Ct.

Can i be sure that my DNase treatment was efficient enough? is there still DNA contaminating my RNA???

thanks

Edited by moljul, 26 May 2009 - 02:38 AM.

[AquaPlasmid]

It's a little tricky with DNaseI digest as most DNaseI have RNase activity at high concentration. Using minimal DNaseI and incubating at room temp for 40-60min could reduce RNA loss. If you check the DNaseI digested RNA sample in a gel, you will know if the digest goes well or not. If the DNA smear is <250 bp, you would most likely have a negative amplification in -RT reaction.

[lsek]

Hi

In real time PCR, it's advisable to include an rt control (DNaseI digested RNA + 1st strand cDNA synthesis components except RTase) in addition to NTC. That way you'll be able to troubleshoot if the signal is due to gDNA contamination.