I have been trying to amplify one lncRNA not very well annotated (has two transcript variants in ensemble and one in NCBI. All there overlap and one has intron as per ensemble). Ct value for lncRNA is around 30 to 35 depending on cell line (comparatively 18s gets Ct value in same sample around 18). My RACE cDNA is fine as I see control beta actin 5 or 3 RACE is working just fine. I am using Clonetech's Smarter RACE kit. Any inputs will be helpful.
Many, possibly most, lncRNAs are not polyadenylated. These will not work with the traditional 3' RACE. 5' RACE should work just like normal. However, we find the effectiveness of RACE to be very dependent on the concentration. Thus, if there are steps like size-fractionation, rRNA depletion etc that can be applied first without degrading the RNA they might help. If you want the 3' end, there is an old method of using polyA polymerase to add an A-tail or even a C-tail, to allow priming starting from the 3' end. It is certainly technically more demanding, however.
Wrong - most lincRNAs are polyadenylated :)
We sequenced 99% of them on PacBio years ago, but never analyzed or published the data. Yes, some of lincRNAs are below 1 rpkm, 250M long iLLumina reads per single sample are not enough to call them.
The tissue abundance is a critical issue; I had these high Cts for a certain cell type. When I checked on UCSC and tried another tissue where it was seeming highly expressed I got a super thick band on my gel.
So,you might need to do a tissue panel and then to do the RACE where it is indeed expressed - otherwise it is always hard with low abundance templates. Of course, they are not always expressed in the same manner across all tissues. So tissue specific expression should also be kept in mind. Had satisfying results with both Invitrogen & Takara.
All the best.
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