We need to assess the levels of six miRNA in human cell lines. We have used miScript primer assay from Qiagen sone time ago, but the primer sequences are not disclosed (universal primer and specific miRNA primer). Now we would like to order primers from a local supplier, instead of ordering it from Qiagen. So the question is: does anyone now where to get these sequences? Or how do they usually design the specific primer?
Specific primers are fully complementary the mature miRNA sequence. Universal primers are not disclosed but you could sequence your PCR products and find it out for yourself.
Thank you, Gracjan. I was think about sequencing, at least I can figure out 5' ends. Do you think that universal primer sequence stops right before oligo(dT) tail?
No, we order primers from qiagen, we decided it will be more reliable and faster
the problem is we do not know where the universal primer ends, but of course you can play with that and optimize the primer sequence and qPCR reaction efficiency, but it may take some time and efforts
Based on the kit supplied by Qiagen and its schematic explanation (see attachment), I would say that the miScript primer assay is identical to the sequence of miRNA (not complementary to it). Did you tried the complementary one ?
Thanks
How did you see this. Is this chart indicate that the miScript provided by QIAGEN is similar to the miRNA sequence ?
Thanks
Mohamed, Forward primer seems to be identical to miR sequence (at least I found some articles where people used exactly the sequence of miR as a primer, but I'm not sure if it will work out for any miR). But the point is that the reverse primer (universal pimer) anneals to sequences from universal tag, which are not disclosed by qiagen/
From an experiment that I did few days back, I ordered a primer for miR-155 which was similar to the miR sequence but on the DNA level, meaning each U will be T and , it worked nicely giving higher CT values compared with the one provided from Qiagen.
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