RNA isolation / purification Bacteria - Gram negative Pseudomonas aeruginosa

Generally isolating RNA from Gram-negative bacteria is easy, however keeping your working environment clean and RNase free (use RNase inhibitor) is essential. Some common points to keep in mind: a) Use fresh samples for isolation or store them by freezing in RNA stabilizing buffer until use. b) Choose the bacterial input amounts carefully, to ensure buffer volumes are adequate and not to overload the columns.

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4 years ago

4 years ago by Paul G. Macon United States

Some help with RNA isolation using Trizol

Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?

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Protocol tips
The total RNA was isolated using the illustra RNAspin kit™ (Ge Healthcare, UK) and ribosomal RNA depletion was performed using the RiboMinus™ transcriptome Kits (TermoFisher Scientific), followed the manufacturer's recommendation. The integrity of RNA was checked in agarose gel (1.5%) in a denaturant condition and the amount of RNA was determined using a spectrophotometer (Figures S7, S8) (BioTek Sinergy HT).
Upstream tips
Pre-treatment with Lysozyme (40 ug/ml)
Protocol tips
To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
Include DNAse treatment for 15-20min.

Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.

Use water to elute the RNA that is warmed to ~60`C
Protocol tips
Following RNA isolation from the P. aeruginosa and S. aureus co-culture, the samples were subjected to rRNA depletion using the MICROBExpress or Ribo-Zero kits. Bioanalyzer electropherogram traces revealed that, while rRNA was reduced upon treatment with the MICROBExpress kit, little to no traces of rRNA were detectable upon treatment with RiboZero
Protocol tips
RNA extraction was done with cell pellets containing 1 × 109 cells from three independent biological replicates of each condition using three different kits/reagent: TRI Reagent, RNeasy Mini kit and Phenol-free total RNA purification kit.
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