Hi,
I wish to validate NGS data of miRNA sequencing from human plasma samples.
The obvious way to do it is of course Taqman, but there are two different assays: the 'traditional' one in which you do RT for each gene; and the 'advanced' in which you make one RT after polyadenylation at 3', 5' adapter ligation and pre-amplification.
The advantage of the first assay is that it is more 'different' from NGS and therefore could be better for validation, but on the other hand it is more labor intensive and you use more material to prepare the RT (the same amount you would use for advanced assay multiplied by the number of genes you want to quantify).
I am inclined to use the advanced assay which has an obvious advantage especially when working with samples with such a low RNA input. My only conecrn is that it is quite similar to NGS in that it uses adapter and also a pre-amplification step. From the pre-amplification step the method is different than NGS becuase it uses a probe and is therefore more biased.
What is your opinion?
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