RNA isolation / purification Bacteria - Gram negative Vibro cholerae

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
Total RNA was isolated from V. cholerae cells grown aerobically to mid-exponential phase (optical density at 600 nm [OD600] = 0.3 to 0.4) in LB medium at 30°C. Briefly, overnight-grown V. cholerae cultures were diluted 1:200 in fresh LB medium and were grown aerobically at 30°C with shaking at 200 rpm to an OD600 of 0.3 to 0.4. The cultures were reinoculated (1:200) into fresh LB medium until the OD600 reached 0.3 to 0.4. Aliquots (2 ml) of the cultures were collected and centrifuged for 2 min at room temperature. Cell pellets were immediately resuspended in 1 ml of TRIzol reagent (Invitrogen, Grand Island, NY) and stored at −70°C. Total RNA was isolated according to the manufacturer's instructions. To remove contaminating DNA, total RNA was incubated with a Turbo DNA-free kit (Thermo Scientific, Grand Island, NY) and concentrated using an Isolate II RNA micro-cleanup kit (Bioline, Taunton, MA).
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"
Upstream tips
For lysis/cell disruption check the protocol beforehand to see which reagents/lab materials are needed
Downstream tips
Minimal DNA is left over using this kit, however if DNA is undesired an additional RNase free DNase treatment is warranted.
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