RNA isolation / purification Bacteria - Gram positive Streptococcus pneumoniae

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 could be the 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
Genomic DNA and total RNA were isolated from S. pneumoniae strains using a Wizard genomic DNA isolation kit and SV total RNA isolation system (Promega), respectively, following the manufacturer's instructions, except that cells were incubated with 0.1% deoxycholic acid (Sigma) at 37°C for 10 min before extraction. RNasin (0.5%; Promega) was added to extracted RNA to prevent it from degradation. cDNA was derived and amplified from RNA using an Access reverse transcription-PCR (RT-PCR) system (Promega) and target-specific primers.
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
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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