I plan to extract mRNA (from total RNA) from fetal sheep organs for transcriptomic work. I am interested in peoples views regarding the efficiency/efficacy of either the direct method, using beads to extract the mRNA based on the poly A tail or the minus method, removing the ribosomal RNA and leaving the mRNA. I am particularly interested in those who have tried both methods. Any help would be appreciated.
Hi Dr. Smith
Both the methods have some pros and cons...the method cane be selected based on your priority:
1) OligodT based purifications of mRNA will result in purifications of mRNAs with polyA tails but you'll loose many other known mRNA with no poly A tails and also many other for which we still don't have any information about the nature of their transcript.
2) The removal of large RNAs (including rRNA) will remove most large RNAs (that may also include som large mRNA transcripts) but will keep many mRNA transcripts irrespective of their polyA status
SO your method of choice should depend on the type of experiments/ type of genes you are trying to analyze....you want to keep most mRNA having polyA tail ( and afford to loose withou polyA) ...or you prefer to have some other RNAs also with mRNA having polA or no poly A tails.....
if you plan to do RNA-seq I would recomment that at least 3 samples are 3' captured on oligo-dT (3' bias) and 3 samples are rRNA depleted (Ribo minus kits)(5' bias); thus, at least 6 samples per point will give a base for statistic and more important to compare for bias.
In the transcriptome work you should be interested for the full length mRNA, which are mostly capped with polyA. I think you should go with oligo dt qiagen column. I have good experience with that also.
Thanks for the responses, while the oligo dt columns sound best I have some concerns over the poly A tails in fetal germ cells, although this may not affect the outcome it does give me some reservations. In short I will try both methods and assess the quality/quantity of RNA recovered and then make a decision.
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