How To Design A Long Noncoding Rna Detection And Quantification Experiment
I am planning on an experiment for detection of novel linnoncoding RNA as well as expression of known lincRNA in patient samples. I was wondering what will be the best approach to extract non coding RNA. I hav eseen Ribo depleted, Ribo zero Hexamer. I sthere a study comparing existing methods of studying such non coding RNA or suggest any best method. I would appreciate if some one in my situation can share their vies, what they have tried and their working experience on which approach one may be best. Thanks
1 answer
Based on recent studies, it is good to use polyA+, long reads (>70 bp), strand specific RNA-Seq to identify lncRNAs. Briefly, you can follow these steps.
- De novo assembly of your RNA-Seq with out using known genes annotation after mapping.
- Remove assembled de novo transcripts that overlap any known transcripts.
- Calculate coding potential by scanning conserved ORFs (open reading frames) and remove the ones which look like coding.
- To further strengthen your lncRNA, overlap H3K4me3 peaks with promoters of lncRNA and H3K36me3 peaks with the gene body.
- Remove transcripts with less than 2 exons
- Remove transcripts that are smaller then 200 bp
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Is your question bioinformatic related?
I think seqanswers would be a better place to ask this. In my opinion we can leave it open because we are nice (and some bioinformaticians might know more about sequencing than a lot of biologists), but otherwise the question is obviously not sufficiently related to bioinformatics, other than that the analysis implies that bioinformatics tools will be need.
I believe researchers may have dealt with it before analysis and can provide better perspective than just wet lab people. However I do agree that it is not a pure programming question Thanks