I want to check the miRNA levels and miRNA transcript is short in length. How to design primer for a specific mature miRNA?
Most of the mirna are of same lenght as of primers so it is very much difficult to design.you can do mirna profiling using qpcr or microarray(professional services).
It is hard to design primers for miRNA because its short length. When using miRNA I found the range of products from Applied Biosystems very reliable. They have primers for mature and for pri-miRNAs. For mature, the innovation they use is a stem-loop primer that avoids the possibility of picking the immature forms and warrants that only mature form is detected. You can check this in Applied Biosystems' webpage or in found a reference in our paper.
http://breast-cancer-research.com/content/14/3/R77
This is a common question on RG, so if you search (e.g. "question miRNA primer"), you will find numerous previous discussions. Martin Rivas correctly points out the length issue and the widely used ABI assays. For details on the stem-loop RT primer approach, see this NAR paper by Caifu Chen https://www.researchgate.net/publication/7455286_Real-time_quantification_of_microRNAs_by_stem-loop_RT-PCR
Other approaches that have been reported to make a mature miRNA sequence long enough for PCR include ligating adapters or extending the miRNA by adding a poly-A tail.
Article Real-time quantification of microRNAs by stem–loop RT–PCR
Hi there,
generally you have two options based on: 1) RNA hybridization, and 2) RT-PCR. First approach seems to be used more often (lots of papers describing the methos), whereas the PCR-based assays are (?) an alternative for it. If you want to compare miRNA level using the northern, design the primers complemetary to the target miRNA. To increase specificity of the hybridization, your oligo-probe may contain some LNA.
If yoyuwant to estimate miRNA level using RT-PCR consider thoes modification of standard RT-PCR protocol:
-digest resulted RNA samples with DNase;
-perform pulsed reverse transcription as described: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225395/
-consider using the universal probe library technology http://www.roche-applied-science.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_2_07_p12-15.pdf.
Good luck!
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