oligos as synthetic "miRNA" for quantification? - would an oligo / primer work in AB RT kit? (Jan/23/2007 )
Hi everyone
I am using the AB taqman micro RNA assays to quantify the level of a particular miRNA in different tissues.
They have published a paper http://nar.oxfordjournals.org/cgi/content/full/33/20/e179 where they used synthetic miRNAs to quantify the number of copies per cell, based on a standard curve generated from RT of known quantities of the synthetic miRNA.
Firstly, does anyone know what synthetic miRNAs are??
Secondly, does anyone think that the stem loop primer in the AB microRNA kit would be able to reverse transcribe an oligo instead of buying in the presubably expensive synthetic miRNA.
I am well aware that a primer would not work as a miRNA.
Anyone got any ideas??
I am using the AB taqman micro RNA assays to quantify the level of a particular miRNA in different tissues.
They have published a paper http://nar.oxfordjournals.org/cgi/content/full/33/20/e179 where they used synthetic miRNAs to quantify the number of copies per cell, based on a standard curve generated from RT of known quantities of the synthetic miRNA.
Firstly, does anyone know what synthetic miRNAs are??
Secondly, does anyone think that the stem loop primer in the AB microRNA kit would be able to reverse transcribe an oligo instead of buying in the presubably expensive synthetic miRNA.
I am well aware that a primer would not work as a miRNA.
Anyone got any ideas??
Two methods for relative quantitation were covered in a previous topic both are relatively cheap details and links here http://www.protocol-online.org/forums/inde...showtopic=21261
Basically extract RNA and polyA tail everything then anneal an anchored PolydT with unique sequence, followed by RT (mRNA and microRNAs are converted to cDNA) then you amplify with specific primer for your miRNA and primer for the unique sequence.
For relative quant (not possible with AB system) you design a second primer for housekeeper gene other micro etc. and use the same cDNA sample again with primer for the unique sequence.
Use Syber to measure both and as its relative no worrries with sample variabliity
According to paper of Raymond et al. (RNA, 2005, 11:1737-1744)
DNA oligo yield Ct values that are identical to or 1~2 Ct values lower than RNA oligos in primer extension based RT-PCR.
As to ABI's loop RT-PCR protocol, it is not sure for somewhat the secondary structure (loop) might play additional role in primer extension efficiency as well.