I have extracted RNA from S. epidermidis using RNeasy mini kit (Qiagen). The extracted RNA concentrations ranges between 23-47 ng/µl per 40 µl . Are these concentrations sufficient to do RNA sequencing or I have to extract more.
I think is also relevant you check the integrity of your RNA in a gel, probably this is more important than taking care of the absorbances. If your RNA do not shows degradation is what matters the most for an appropriate library preparation I believe.
That should be enough RNA in terms of quantity if you are preparing total RNA libraries using a kit such as Illumina's Total RNA Sequencing preparation kit (which requires 0.1 to 1 microg total input). You may or may not have to concentrate your samples first, however.
Thank you. I am having only 0.9 mcg/40 mcL. Is this enough? Also i have to do rRNA depletion using illumina Kit. In addition, the quality was checked with nano´drop and the results are attached for your ease reference. The ratio of 260/230 got decreased and I got slight increase in the ration of 260/280. These contaminations may affect the RNA seq. How can i rectify this problem?
I think is also relevant you check the integrity of your RNA in a gel, probably this is more important than taking care of the absorbances. If your RNA do not shows degradation is what matters the most for an appropriate library preparation I believe.
Nanodrop is not very precise for low RNA concentrations, and as written above, I would warmly recommend to check your RNAs with some precise electrophoresis device, such as Agilent BioAnalyzer. It will give you an idea of the integrity of your RNAs and of their quality. I don't know how you plan your sequencing (do you do the libraries yourself or do you rely to an external / commercial provider / facility?), but usually sequencing facilities perform this step before making libraries from your RNAs. If you're interested only in mRNAs (not miRNAs or ncRNAs), I would not recommend rRNA depletion, but rather polyA RNA selection, it works better. I am not too concerned about the A260/280 and A260/230 ratios, the first one is very good to good for all your samples and the second one mainly reflects a guanidium thiocyanate contamination from the RNA extraction kit, that'll be washed during the libraries preparation steps (I several times made RNA-seq libraries from samples with quite low A260/230 ratios without any further troubles). Good luck!
Yes, I do agree that the NanoDrop Spectrophotometer doesn't give accurate or reliable results with low RNA concentrations. I myself experienced that phenomenon several times. I will also suggest that you double check the integrity of your RNA by electrophoresis as suggested by other experts. You could extract more RNA or not, depending on your interest.
For TRUseq total RNA libraries, 100 ng of total RNA is usually enough. As recommended by others, I would definitely recommend using a bioanalyzer to find the RIN value to quantify the RNA integrity. Moreover, I would NOT recommend using a low input library preparation if you can possibly avoid it, due to the amount of technical noise that can be introduced through the preliminary amplification strategies. See this article for additional information: Article Technical Variations in Low-Input RNA-seq Methodologies
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