I am trying to figure out the specific miRNA which is actually involve in splicing of my target gene mRNA. I want to ask what criteria should i follow. ( i mean choose 1-3 among many)
secondly, if the miRNA is edited using any of tool such as CRISPR Cas9, what aspect should be kept in mind, as the mutation can be caused not only in miRNA i selected, its can cause some where else also as the miRNA family contain some how similar not completely different sequence.
How to chose a potential miRNA of interest see for ref. current lit. (via Pubmed), e.g. Saliminejad K, Khorram Khorshid HR, Soleymani Fard S, Ghaffari SH. J Cell Physiol. 2019 May;234(5):5451-5465. doi: 10.1002/jcp.27486. Epub 2018 Nov 23.
Theoretically speaking, synthetic miRNA oligos or generated through vectors has two possibilities; transcription silencing or translation inhibition. You can generate miRNAs using vector (pSilencer or pRNAT) by selecting 21-25nt imperfect complementary strands with loop i.e., not exact complementary strands. Incase of endogenous miRs, non-complementary strands acts for translation inhibition by binding to target binding site at 3' UTR of mRNA. Incase of complementary strands, they promote transcription inhibition by cleaving the mRNA, which is similar to siRNA mechanism.
In your case, you wanted to generate gene silencing via transcriptional silencing. So you can use siRNA vectors which will provide you silencing of your target gene.
miRNAs are identified from sequence data uploaded or entered by the user. Sequencing data could be derived from a variety of sequencing projects. For instance, in a comparative sequencing project, miRNA expression in tissue samples could be examined with and without a transcription factor silenced by siRNA.
Dear Prof. Bashdar Mahmud Hussen thank you for your contribution
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