Hello, does anyone have an idea why I have such a huge amount of tRNA and 5S rRNA ir the sample of E. coli total RNA?Agilent (A) and agarose gel electrophoresis (B) results as well as RNA purity measurements acquired with NanoDrop (C) are provided in the picture)
For total RNA isolation bacteria were grown in LB medium at 37C for about 6 hours – until it reached early stationary phase. At that point OD600=5,41 of bacteria was mixed with STOP solution (95% ethanol and 5% phenol) at a ratio of (parašyk santykį 8:1). Cells were spun down at 1 800× g, 4 °C, 20 min and kept at -80 C for a month. Frozen cell were ground to a fine powder in liquid nitrogen and about 100 mg of it was used for a total RNA extraction using RNAzol reagent according to the manufacturer's protocol. RNA pelet was solubilised i 40 μL of DEPC H2O, aliquated, flash frozen in liquid nitrogen and kept at -80 C for further analysis.
The results provided were basically the same for as many as 24 samples of wild type and couple of mutant E. coli strains. As I have never seen this - such a huge amount of tRNA and 5S rRNA fraction - before, I wonder maybe you could help me with explaining such a phenomenon and with recommendations if the total RNA I obtained can be used for Norther hybridization analysis? Can I hope, that all RNAs of different sizes are represented equally in such a sample?
We had the same problem. And we found out that this depends on the growth phase. The more you are in the stationary phase, the more 5S and tRNAs you can find. Just try out what you see if you isolate total RNA in the exponential growth phase.
It is because you isolate your RNA during the plato phase of culture. In this phase they stop diving and start to produce secondary metabolites. This process requires a lot of tRNAs.
Anika Wahl and Enes Yağız Akdaş thank you very much for your answers! Indeed, we tried to purify total RNA from several phases and we saw the differences.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays