RNA isolation / purification Bacteria - Gram positive Staphylococcus saprophycitius

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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4 years ago

4 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
For RNA to dissolve better and to yield high levels, preheat elution buffer at 95C.
Addition of β-mercaptoethanol in lysis buffer can be skipped because the phenol will do the job of nuclease inactivation.
To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.
Do vortex for 2-plus minutes. This will facilitate the removal of tightly bound proteins typically associated with RNA.
Protocol tips
Total RNA was isolated from frozen tissue using the Promega Total RNA Isolation System (Promega, Madison, WI, USA). For cDNA synthesis, 4–8 μg of total RNA was reverse transcribed with Superscript III reverse transcriptase using an oligo-(dT)12–18 primer according to the supplier’s protocol (Invitrogen, Carlsbad, CA, USA). A single cDNA preparation from each specimen was used for the assay of all antimicrobial products tested.
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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