I am collecting rare cell types from the nematode C. elegant by FACS for RNA extraction and subsequent sequencing. For some populations we get 5,000 - 10,000 cells. We sort directly into TRIzol LS, then use phase lock heavy gel tubes and isopropanol precipitation, followed by DNase (Zymo) treatment and clean-up with a Zymo Clean-up and concentrator kit. Our RNA extractions have been highly variable in yield and quality. In optimizing the protocol, I have found that leaving out the DNase Treatment, I can reliable and consistently get RNA out of as few as 7,500 cells. The RNA concentrations range from 300 - 3000 pg/uL (as measured by Bioanalyzer Pico chip). The average RIN is around 7.5 - 8.0. DNase I treatment of the samples almost always results in decreased quantity and RIN values. I've posted three images of Bioanalysis profiles from the same samples before (left) and after (right) DNase I treatment. Any help on how to avoid this loss of quality and quantity would be gratefully appreciated!
What kind of RNA-Seq do you perform? Do you analyze mRNA? For this, you have to get rid of rRNA that constitutes >90% of total RNA, this step is called rRNA depletion. You also can use enrichment to separate only specific RNAs (the most common example is mRNA enrichment using magnetic beads conjugated with oligo-dT probes so they hybridize with polyA region, and only the processed mRNA molecules would be kept). Do you know which depletion/enrichment method you would use? If you use some form of enrichment so you only separate RNAs that you need, you probably wouldn't need DNAse step at all because DNA molecules would be discarded as well as RNAs that were not selected?
We have most commonly used oligo-dT selection, but we are interested in also using this RNA for small RNA seq if possible. And from what I've read, if the genome has polyA repeats, there is no guarantee that polyA selection will exclusively yields from mRNA, though it will likely enrich for those. We've also looked into rRNA depletion, but the methods our lab has tried in the past did not work for our model organism.
I see. And do you absolutely have to use TriZol for RNA extraction here? It is the cheapest method that yields the maximal amount of total RNA you can extract, but it is also the least pure method. Can you afford special columns designed for minor RNA quantities? Although you have to check if these columns are good for small RNA as well, most columns are not.
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