ABI Taqman Low density microRNA Array card - (Sep/21/2009 )

Hi There,

I work on miRNAs. has anyone here used ABI Taqman low density miRNA array (human array A) for miRNA profile study? I did two samples using the cards and found around half of the miRNAs were not determined (the card contains 377 microRNAs, only 190 miRNAs were determined). CTs for U6 and RNU44 were alright (17 and 22 respectively). my sample is total RNA isolated with mirVana kit followed by RT and preamplification. has anyone met this problem before? If you have experience with TLDA card, please help. Thanks.

LW

What did ABI tech support have to say? Usually they're really helpful.

how much RNA did you use to perform RT? i used 10ng in 15ul reaction and i didnt do the preamplficiation and it worked fine. i heard from the salesman of ABI the preamplficiation got some problems before, and the company later modified the protocol a bit. i am not sure if your problem is because of the preamplificiation step.

samantha

Hi LW,

I have been working on the ABI taqman arrays (used over 50). I think it is normal that 190 miRNAs are undetermined in the A array. It is a screening process and I have similar results. Expect even more to be undetermined in array B (possibly getting a flagged plate for well percentage amplification in SDS manager). Also remember, over 35 cycles is classed as not expressed in these arrays!

I am currently having some issues validating my results. This is because the cDNA conversion in the megaplex reaction is over 40 cycles, whereas for a single assay it is only a single conversion. This makes it difficult validating because miRNAs come up later in the cycles for single assays. In the middle of getting help from tech support about this issue.


RNA (that's actually my initials)