This method uses the Agilent Total RNA Isolation Mini Kit to isolate high-purity RNA from yeast cells. The method has the advantage of removing cellular contaminants such as proteins, carbohydrates, and genomic DNA, resulting in a purer RNA product. The method uses a carefully prepared yeast spheroplast followed by complete homogenization of the sample to remove high levels of cell wall components and other proteins, thus achieving high efficiency in the separation of yeast RNA. Compared with other commercial methods, the Agilent method yields comparable yeast total RNA yield and contains significantly less genomic DNA than RNA isolated with a competitor's silica-based kit. Agilent 2100 Bioanalyzer analysis showed that the isolated RNA had high quality and integrity.
This document details a method for isolating high-purity total cellular RNA from yeast cells using the Agilent Total RNA Isolation Mini Kit (p/n 5185-6000). The method is a phenol-free, spin-column technique that employs a unique prefiltration column to remove cellular contaminants, including genomic DNA, without the need for DNase treatment. The protocol involves the preparation of spheroplasts using a lyticase-containing buffer to degrade yeast cell wall components before lysis and extraction. Analysis using the Agilent 2100 Bioanalyzer demonstrates that the isolated RNA is of high quality and integrity, with significantly lower levels of genomic DNA contamination compared to silica-based methods.
| Kit Part Number | 5185-6000 |
|---|---|
| Incubation Temperature for Spheroplasts | 30 °C |
No, the kit removes genomic DNA using a unique prefiltration column without the need for DNase treatment.
The yield is approximately the same as silica-based methods, though it contains significantly less genomic DNA.