RNA isolation / purification Bacteria - Gram positive Mycobacterium tuberculosis

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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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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PureLink ™ miRNA Isolation Kit

Thermo Fisher Scientific

Protocol tips
For retrieval of RNA from the extraction solvent following mechanical disruption, we compared simple isopropanol precipitation of RNA to solid-phase isolation of RNA using the Purelink miRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA). A summary of all conditions tested and their respective nucleic acid yields before DNase I treatment as assessed by UV spectroscopy is detailed in Supplementary Table S1; the analyses were performed in three biological replicates.
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
Kit relies on bead-beating and magnetism, so adequate lab equipment is required, check the manual for required materials.
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.
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