miRNA QPCR advice - taqman (Mar/08/2007 )
Just wondering if anyone has any advice -
Working on mouse guts - which as i mentioned in an earlier post are not compatible with the mirVana kit.
at the moment i am using trizol up to collection of aqueous phase - i then add ethanol and swtich protocols put through the mirVana columns. my rep suggested that the straight trizol would allow too many inhibitants to enter the RT and QPCR... (but then it was the ambion rep!)
i am struggling with the real time though -quite variable results. I'm doing 3 RTs per sample and then 4 QPCRs per RT (ie. lots!) but there is a fair bit of variation. we normally clean up cDNA and standardise conc to 30ng per well for a normal transcript.
the taqman assays say just dilute cDNA a min of 1:15 then use - it seems a bit vague to me - does everyone do this?
i have also tried the mermaid kit to clean up small dnas prior to the taqman run but it still seems a bit variable
i am trying to confirm some aCGH results - but my taqman is the opposite about 50% of the time!!!!!!!!!!!!!!!
thoughts? am i being overly concerned? should i just accept the data i am generating????
I feel Trizol reagent is good enough for RNA purification, if you still need further purification, it is better used purified RNA on mirVana columns...
Another suggestion is try Trizol purified RNA, but diluted it by water before RT and QPCR, which might reduce the possible inhibitants effect
Hi, istarted using PureLink miRNA isolation kit from invitrogen. it works quite well, but if yuo use Trizol instead of the vendor's lysis buffer it works better! Anyway now i use just Trizol extraction doublin the RNA amount when retro transcribing. I have no contaminants and clean results with taqMan miRNA probes from Applied Biosystems.
I also tried the Ambiion system, but found that Trizol and invitrogen's kit are superior.
Hope it helps!
we use 10 ng of total RNA per RT reaction