In my lab, a target gene was knockdowned (RNAi). Both RNAi and Control samples were separately prepared to construct libraries of small RNAs that were deep sequenced using Illumina platform (36 cycles). The sequencing results (performed in triplicate) showed a clear differential pattern in the length distribution of reads when Control and RNAi samples were compared. Most reads from RNAi samples ranges from 10 to 17 nt and many of them are rRNA. On the other hand, while reads generated from Control samples are longer than 20 nt. What these differential distribution patterns mean? How to explain the high abundance of ribosomal RNA in the RNAi samples and low abundance in Controls? Have you similar results and/or advices on how to interpret these data? Do you know any article discussing it? Please, share your expertise and beliefs.
Hello Francis,
Have the knock-down and control samples been prepared only once (followed by three sequencing runs on the same pair of samples)? Then it could be an accident, but if you had three pairs of independent transfection reactions followed by three pairs of RNA preps, then I am at a loss. A few things I would think about:
How do you isolate/prepare RNA for library construction? Have you checked general RNA quality before library construction, or did you start out with only short RNA?
For information about RNA quality assessment, please take a look here: http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/rna-isolation/tech-notes/assessing-rna-quality.html
One more question: what was your control? I would suggest using a si/shRNA with scrambled or unrelated sequence.
Good luck,
Vera
Thanks for advices. Different RNA quality tests were used: gel electrophoresis, purity, RIN. I'm leaning towards the unintended degradation due to off-targets effects. What are your thoughts?
Hello Francis,
I wouldn't be surprised to see more ribosomal RNA in your RNAi samples. Depending on the target gene silenced, many other genes and pathways will be activated, which will naturally require more ribosomes for protein production.
No clue for the difference in size... sorry.
Hope it helps!
Jayme
Thank you all for reply. The last answer was sent in April 2013. So, please, would you like to share updates on this topic? We could discuss not only RNAi vs. RNA-Seq but also CRISPR/Cas9 vs. RNA-Seq. Hope your contributions and advices.
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