RNA isolation / purification Bacteria - Gram positive Streptococcus pyogenes

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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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

RNeasy Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	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"

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

Direct-zol RNA Kits

Zymo Research

Upstream tips	Protocol tips	Downstream tips
Tough-to-lyse samples (i.e.,gram-positive bacteria, yeast, fungi, animal tissue, plant, etc.) can be homogenized in TRI Reagent® (supplied with kit) with a mortar/pestle, dounce, tissue grinder, or bead beating with a high-speed homogenizer prior to RNA Purification.

Upstream tips
Tough-to-lyse samples (i.e.,gram-positive bacteria, yeast, fungi, animal tissue, plant, etc.) can be homogenized in TRI Reagent® (supplied with kit) with a mortar/pestle, dounce, tissue grinder, or bead beating with a high-speed homogenizer prior to RNA Purification.

Manufacturer protocol Publication protocol 1 Relevant paper

TRIzol™ LS Reagent

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
TRIzol LS reagent is intended for liquid samples, using solid samples will decrease RNA yield

Upstream tips
TRIzol LS reagent is intended for liquid samples, using solid samples will decrease RNA yield

Manufacturer protocol Publication protocol 1 Relevant paper

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