RNA isolation / purification Bacteria - Gram positive Lactobacillus amylovorus

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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Protocol tips
Total RNA was extracted from the lysates by an InviMag Universal RNA Kit (Stratec) using the KingFisher Flex (Thermo Scientific, Langenselbold, Germany) according to the manufacturer’s instructions. The yield of total RNA was estimated spectrophotometrically at 260 nm and isolated RNA was stored at -80°C. RNA quality and integrity was verified using the Agilent 2100 Bioanalyzer (RNA 6000 Nano Chip, Agilent, Waldbronn, Germany).
Upstream tips
Be careful to create an RNase-free working environment
Protocol tips
Always mix the sample tube well after addition of each reagent.
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