I've just got the results of RNA QC for RNA-seq using mouse brain
tissues and there turned out that many samples didn't have enough
total RNA (19 out of 24 samples, which means 5 samples had
enough RNA).
Other than that RIN value and rRNA ratio were above the criteria.
In this case, what would be the main problem for the low RNA
quantity?
The amount of tissue sample itself? or do I need to care
more for the breakdown of RNA by RNAse?
Thank you.
Were your RINs for all samples good? If that is the case degradation of RNA was not a problem. You would have to increase the amount of tissue to increase RNA yield. Good luck!
How do you isolate your RNA? We sometimes have concentration issues using column kits. Have you tried different methods?
Hi,
You can try to make the isolation again (from the appropriate amount of input material, I can suggest mirNeasy from Qiagen (but do not skip DNAse treatment)), but if you have same problem again and if the quality of your samples is good (RIN), you can just normalise the amount of RNA for all samples to the lowest sample and use an appropriate library prep kit. There is basically no limitation in the minimum input amount - you can do RNA-Seq starting from roughly 10 pg of RNA (1 cell).
Best,
Michail
What where the obtained quantities ? As said above, RNA-seq is done from single cells, so there is basically no limit for starting quantities. The only limitation is cost, as library prep kits have a tendency to be more expensive for lower quantities. If you use a external service provider, they often propose to use single cell kits for small quantities from low number of cells. Good luck!
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