RNA isolation / purification Bacteria - Gram positive Bacillus anthracis

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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Upstream tips
Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample.
Different samples require different methods to achieve complete disruption.
Incomplete disruption results in significantly reduced nucleic acid yields.
Overloading the spin columns significantly reduces nucleic acid yields.
Protocol tips
Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption.
Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids.
Excessive homogenization, on the other hand, results in shorter genomic DNA fragments.
The centrifugation temperature should be 20–25ºC.
Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.
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"
Protocol tips
If the RNA yield is low, repeat RNA elution, but incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging.
All centrifugation steps should be performed at 15–25°C except for phase separation
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