RNA isolation / purification Yeast - Schizosaccharomyces pombe

Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.

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Found 3 matching solutions for this experiment

TRI Reagent® Sigma + NucleoSpin® RNA

TRI Reagent® Sigma

Sigma-Aldrich

NucleoSpin® RNA

Macherey Nagel

Protocol tips
total DNA was liberated from cells by heating their suspension in water for 5 minutes at 95°C and removing cellular debris by spinning at 13200×g for 3 minutes at room temperature. For total RNA preparation, cells were pulverized in liquid nitrogen and RNA was isolated using TRI Reagent (Sigma) followed by purification with NucleoSpin RNA Clean-up (Macherey-Nagel). For total cDNA preparation, 2 µg of total RNA was used with RevertAid M-MuLV Reverse Transcriptase (Fermentas) according to the manufacturer’s recommended protocol.
Protocol tips
The following might help to increase RNA yield
- Heat the DEPC Water to 70°C before adding to the column.
- Increase the incubation time to 5 minutes.
- Increase the elution volume.
- Repeat the elution step with fresh DEPC Water (this may increase the yield, but decrease the concentration).
- Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).
Downstream tips
Isolated RNA was treated with RNase-free DNase I
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.
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