The size miRNA is 89 bps and the primers I have designed (using traditional method for designing gene primers) should give an amplified product of 71 bps. But I am getting product of higher molecular weight. What should be done? Also I have seen that usually people add a stem loop to miRNA (probably to increase its length), how a stem loop could be designed?
Hi Saadiya
Higher products means that your primers do not map at the right sequence, or not specific enough to get the right amplification.
https://genome.ucsc.edu/cgi-bin/hgPcrtry to do an in silico PCR to see what's wrong (https://genome.ucsc.edu/cgi-bin/hgPcr).
all the best
fred
Designing highly-specific primers for microRNAs are not as straightforward as (coding) gene primers. There are commercial kits that help modify microRNAs so that they are more easily amplified after cDNA conversion. There are differing kit chemistry at the cDNA conversion level to consider, it is generally split into two methods: polyA tailing (universal RT) or by the addition of something like stem loops primers. Thermo has an excellent video of these 2 concepts (https://www.thermofisher.com/blog/behindthebench/finding-the-right-microrna-mirna-assays/). If you want to add stem loop primers, look into commercial vendors for a kit. But also consider the polyA tailing method which gives you flexibility. Understand the chemistry of the methods, you will be locked into the particular way of doing things if you choose one. Each has its own advantages & disadvantages. If you are happy with SYBR-green based assays, polyA tailing is not a bad start.
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