Hi
I want to do NGS for microRNA presents in my exosomal samples obtained from cell culture. I have isolated exosomes followed by total RNA extraction. However, when I checked the quality on the bioanalyzer it shows only 1 peak around 25 and did not give any RIN number. I am confused as to whether my RNA sample is ok and it has only small RNA only, or it has degraded. Please give me suggestions as I am very confused.
Hi Atoni,
I think you may have one peak rich for small rand, which should be mainly miRNAs. Enen you have some other rnas such as mrna, it will be difficult to have peak since the size is quite different. In addition, miRNAs should be abundant in exosomes compared to mrnas.
How much quantity is actually required for microRNA sequencing....i get very little total RNA (which is mainly microRNA) from huge cell culture volume. please suggest.
attaching the file
RIN value is calculated based on the ratio of big RNA to rRNA. RIN value is important for total RNA sequencing. However, when you are trying to specifically isolate microRNA population from your samples, RIN value is no longer important. MicroRNAs are very stable, even you can detect them from paraffin tissues. you may read the following article by M. Eldh et al. / Molecular Immunology 50 (2012) 278–286
Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods.
If you used Agilent system for determining RIN in your samples, you have to keep in mind that high amount of small RNAs purified, can have a negative impact on RIN values, since the algorithm behind the RIN calculation assesses these small RNAs as degradation products. So, the RIN and similarly of the RQI(RNA Quality Index) of the Expression system are not the method of choice to quality control RNA eluates containing small RNAs. For the Agilent instrument, a special chip (small RNA Lab chip) system is required that allows a more detailed look at the small RNA species.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA