RNA isolation / purification Yeast - Coprinus cinereus

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

Protocol tips
To confirm the nbs1 gene structure and sequence as predicted by the Broad Institute genome sequence, reverse-transcription (RT) PCR was used on purified poly-A RNA from a J6;5x4 K+6 mushroom (SuperScript One-Step RT-PCR for Long Templates, Invitrogen; PolyATtract mRNA Isolation System III, Promega)
Protocol tips
The first reagent of the kit (Buffer RLC) was added to the frozen tissue, and ground using a mortar and pestle.

Both enzymatic and mechanical lysis can be used for yeast. Enzymatic lysis doesn't require additional lab equipment, but takes longer than mechanical disruption.
TRI Reagent® MRC

Molecular Research Center, Inc.

Protocol tips
Total RNA was extracted by TRI® reagent (Molecular Research Center, Inc) and Poly(A)+ mRNA was furthered isolated using the PolyATract® mRNA isolation system (Promega).
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