I have high peaks in the fast region of my total RNA as seen in the attached pictures. The samples 9 and 12 have different problems, with 12 probably being somehow contaminated and 9 being degraded. But the remaining samples show the very high peaks around 25- 30s which correspond to where I would expect small RNAs and 5s RNA. But they are much bigger than my rRNA peaks. What could be the cause of these and will they pose a problem in downstream applications?
Extraction was done using trizol from gram negative bacteria.
I know that trizol yields a higher proportion of small RNAs then other extraction methods, but this seems like a lot to me.
Thank you for your answers!
It may look like that if you harvested your cells after the culture entered stationary phase. In stationary phase, cells contain much fewer ribosomes. tRNA population somewhat shrinks too but to a lesser extent, and there are many small degradation products of mRNAs and rRNAs digested by bacterial degradosome. As a result, the 16S and 23S rRNA peaks have decreased area as compared to the small RNA peak.
By the way, here is a good illustration of how relative size of peaks on Bioanalyzer proiles changes depending on culture growth phase (E, exponential growth; G, stationary phase):
(taken from https://openi.nlm.nih.gov/detailedresult.php?img=PMC4357692_gku1317fig1&req=4)
Thank you for your answer! I never thought about that. It could be an explanation for the bottom row ( 10; 11 and 12).
Could it also be that my organism is just naturally having an overabundance of tRNAs?
Each Column represents one of my triplicates and the four samples per triplicate were taken during different growth phases. 1; 2 and 3 are from the early exponential phase, 4; 5 and 6 from mid-exponential phase, 7; 8; 9 are from the transition to stationary and 10; 11; 12 are from very stationary cultures.
Well, then it doesn't seem to be the case 'cause I would expect much stronger differences between rows than seen on the figure. It might be because of some metabolic peculiarities of your bacteria, not necessary tRNA overabundance. Something like synthesis of many small regulatory RNAs under certain growth conditions. Trizol might have some effect but it's usually seen at high RNA concentration in samples. Actually, things may become clearer after sequence analysis.
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