RNA isolation / purification Bacteria - Gram negative Salmonella enterica

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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2 years ago

2 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
For RNA to dissolve better and to yield high levels, preheat elution buffer at 95C.
Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation.
To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.
Do vortex for 2-plus minutes. This will facilitate the removal of tightly bound proteins typically associated with RNA.
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
This kit involves a bead-beating step, make sure that you have the appropriate equipment
Downstream tips
When checking the yield by means of a specrtophotometer, use the supplied TE buffer to zero the machine
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