RNA isolation / purification Bacteria - Gram positive Staphylococcus epidermidis

The biggest problem in isolating RNA from gram-positive bacteria is the disruption of the cell wall. A lot of protocols employ enzymatic digestion (pretreatment) which may affect gene expression patterns of certain genes. Therefore physical disruption using beads can be a best alternative.

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1 year ago

1 year 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 following might help to increase RNA yield
- Heat the DEPC Water to 70°C before adding to the column.
- Increase the incubation time to 5 minutes.
- Increase the elution volume.
- Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration).
- Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume)."
PureLink™ RNA Mini Kit

Thermo Fisher Scientific

Upstream tips
For lysis of yeast, extra enzymatic or mechanical disruption is recommended.

For bacteria, lysis using lysozyme is recommended
Protocol tips
Depending on the material you want to isolate RNA from, please refer to the manual for additional reagents/lab equipment that are required
Downstream tips
DNase is not supplied with this kit, a DNase kit is available which is compatible with this kit
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"
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