RNA isolation / purification Bacteria - Gram positive Staphylococcus aureus

The challenge in isolating RNA from S. aureus cells 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 is considered to be the better alternative.

3 Matching solutions
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5 years ago

5 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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Found 3 matching solutions for this experiment

RiboPure™ RNA Purification Kit, bacteria

Thermo Fisher Scientific

4/5 - 2 Reviews - 50% recommends this

Upstream tips	Protocol tips	Downstream tips
- Use of physical disruption of cell walls - Enzymatic digestion of cell walls		When checking the yield by means of a specrtophotometer, use the supplied TE buffer to zero the machine

Upstream tips
- Use of physical disruption of cell walls - Enzymatic digestion of cell walls
Downstream tips
When checking the yield by means of a specrtophotometer, use the supplied TE buffer to zero the machine

Manufacturer protocol Publication protocol 2 Relevant papers

RNeasy Plus Mini Kit 1

Qiagen

4/5 - 1 Review - 100% recommends this

Upstream tips	Protocol tips	Downstream tips
Pre-treatment with lysostaphin	To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	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
Pre-treatment with lysostaphin
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

Manufacturer protocol Publication protocol 1 Relevant paper

MICROBExpress™ Bacterial mRNA Enrichment Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
This kit removes 16S and 23S rRNA so that only mRNA remains. It is not suitable for only RNA isolation. Requires a magnetic stand

Upstream tips
This kit removes 16S and 23S rRNA so that only mRNA remains. It is not suitable for only RNA isolation. Requires a magnetic stand

Manufacturer protocol Publication protocol 1 Relevant paper

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