The LncRNA I am interested in studying has many transcript variants, 14 as per ensembl. I want to study the effect of over-expression of this LncRNA. To do so I want to clone the LncRNA into a mammalian expression vector and temporarily transfect it in a suitable cell line. I do not know which transcript variant is the most abundant, nor do I know which is functional. I would like to i which transcript variant I should clone into the expression vector? Should I clone the largest transcript variant? Should I PCR amplify the entire region from the genome and clone it? Any advice would be appreciated.
Thank you
As a starter I would look at already published lncRNA-seq datasets to identify which is the most abundant variant of this lncRNA.
Aside from looking at public RNAseq data, I would also recommend to design primers for individual isoforms (if that is possible) and measure isoforms on RT-qPCR in your system - though it can be challenging if they are not very different.
As a side note, in my experience, overexpression from a plasmid does not always help capturing the function of lncRNAs, especially those lncRNAs that tend to work in the nucleus. I would highly recommend knockdown or knockout experiments as a complementary and possibly more reliable approach in case you are trying to find out the function of your lncRNA. This way you will probably be able to target siRNAs or antisense oligonucleotides to specific exons of the isoforms that you have.
Good luck!
lncRNAs are very trendy. More importantly, I would also ask why are you studying this paricular lncRNA and what is your assay or readout for its function. As noted above, databases often list every variant or EST ever recorded but these may not be expressed in your cells of interest.
I will suggest you to overexpress by dcas9 VP64 or SAM which will be useful because you are regulating at a genomic level, so that you don't need to worry about the variants. Can you please check lncipedia and also you may know whether it is functionally characterized previously or not.
@nanda thanks for your reply, I considered using VP64 or other transactivators, but they do not always seem to work. The point of binding of dCas9-VP64 requires some screening and its optimisation can take a long time. Even then it may not work as its binding to the promoter is influenced by its accessibility and structure.
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