I am trying to clone a few lincRNAs to express them in cell lines. I can easily quantify them by qPCR. However when I try to amplify the full lincRNA for cloning using cDNA as template, I get no amplification by PCR.
I imagine that this is a result of the tiny concentration of these molecules in the samples, but did you have the same experience? Any suggestions?
I also encountered the same problem. I used two different Taqs. Didn't try the qPCR just yet.
Did you manage to overcome the problem? Do you have any tips?
Hey Kathy
In fact we overcomed some of the problems. But this is a question of luck. In fact lincRNAs are very low-expressed transcripts, so, the sucess depends basically on the choice of the right cell line or tissue to get your RNA. So... I am afraid that there is no magic... sorry
Let me know if I can help. Gooooooood Luck !
Paco
Thank you!!
BTW - which cDNA Reverse Transcription Kit are you using? Do you know if there is one that have more primers to non-coding regions, to enrich ncRNAs?
Kathy
We are using Invitrogen's reverse transcription kit. We have used specific primers in cDNA synthesis to enrich lincRNAs, but as I told you, everything depends on the abundance of the lincRNA in your RNA pool. You can try, but sometimes it will fail.
Best. Paco
Just wondering where to get the gene sequences, cDNA clones or plasmids for these lincRNAs? I tried to look into some databases but could not find?
Hi Alok, I found the sequences in the database are not always correct. In my case, the best way is to do race based on the seq from database like noncode. Then you can start cloning with the 5' 3' infi.
LncRNAs are having higher transcripts, hence cloning them is difficult.
It depends on the length of the transcript. Some of them are less in size (2.3kb) and some of them are too big (more than 10 kb) hence cloning them is difficult, unlike regular coding sequence.
But part of them can be subcloned!
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