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What is the best practise for detection of novel long noncoding RNA as well as the expression of known lincRNA. A litertaure survey showed Ribo depleted, Ribo zero, Hexamer approaches. I wonder if there is a study comparing existing methods of isloating non coding RNA or any best method. It will be great to have feedback if some one has tried varoius methods and their working experience on which approach may be best. Thanks
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You can't simply enrich for non-coding RNA. The method people use is to generate two datasets. Total RNA-seq and Ribo-seq. Then, you need to look for regions that are found in the total RNA-seq dataset, but not in Ribo-seq. -
long noncoding RNA detection and quantification experiment
Thanks Dario1994,
Consider me as new in this area and I am getting little confused with the literature available e.g http://mbio.asm.org/content/1/5/e00206-10.full
suggest that non-ribosomal RNA should give ncRNA? Any thoughts?Comment
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I understand why you are confused now. The issue is that most lncRNA don't have a poly-A tail, so the older method of enriching for RNA in the sample by grabbing them with oligo dT sequences missed a lot of them. Ribosomes are some of the most common RNA in the cell, and they do not have A-tails. You can avoid most of your sequencing reads originating from them by doing poly-A enriched RNA sequencing.
The other approach to RNA sequencing is total RNA sequencing. But, the problem with this is that ribosomes are highly present in the sample. So, you need to do the ribosome depletion treatments to get rid of them. But, with this method, you won't lose the lncRNA without the poly-A tail.
My original point was that by doing RNA sequencing of any kind, you can't simply tell if a new transcript you find in your data is protein-coding, or not.Comment
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noncoding RNA detction
I am also looking for the ways to detect noncoding RNA, Waht is teh ebst strategy to detect and quantify noncoding RNA . Papers have described selective use of Ribo depletion to enrich linc RNA. However it has been suggested just depletion of Ribo RNA may not be sufficient to enrich linNoncoding RNA.
Any suggestion what should be the best way to go - just ribodepleted or both polA vs Ribo-depleted libraries should be best to detect linc noncoding RNA.
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noncoding RNA
I have a very basic question is there a rule what may be good for detection of noncoding RNA (long), -PE or SE longer read in case of ribo-depleted. Is there an advantage/ disadvantage of one over the other. Literature search suggest use of SE/ PE both and even longer SE reads. Any thoughts or experience?Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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