When purifying total RNA from yeast that have grown on nitrogen-limited medium, it always appears more degraded when coming from stationary phase than from log phase.
RNA coming from log phase always looks fine.
I'm using a modified protocol from A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae (Schmitt et al., 1990).
Any recommendations?
Thanks a lot!
The most common and effective method is to use a commercial RNA purification kit. These kits typically use a combination of physical and chemical methods to extract the RNA from the cells and remove any contaminating proteins, DNA, or other molecules.
To purify RNA from yeast using a commercial kit, you will need the following materials:
A commercial RNA purification kit
Yeast cells that have reached stationary phase
RNase-free water
RNase-free tubes and pipettes
A centrifuge
Once you have gathered your materials, follow these steps:
1. Centrifuge the yeast cells to pellet them.
2. Discard the supernatant.
3. Resuspend the pellet in RNase-free water.
4. Add the RNA purification kit according to the manufacturer's instructions.
5. Centrifuge the sample according to the manufacturer's instructions.
6. Discard the supernatant.
7. Wash the RNA pellet with RNase-free water.
8. Centrifuge the sample according to the manufacturer's instructions.
9. Discard the supernatant.
10. Resuspend the RNA pellet in RNase-free water.
11. Quantify the RNA using a spectrophotometer.
12. Store the RNA at -80°C until use.
Following these steps will help you to purify high-quality RNA from yeast that have reached stationary phase. This RNA can then be used for a variety of downstream applications, such as microarray analysis, RNA sequencing, and Northern blotting.
Make sure to use RNase-free materials and reagents throughout the procedure. Work in a clean environment to minimize contamination. Follow the kit instructions.
Do not exceed the recommended incubation times or temperatures. Precipitate the RNA as soon as possible after purification to prevent degradation. Store the RNA at -80°C/ -112°F until use.
Source: https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction.html (April 10, 2023)
Dear Diana Villarreal-Huerta
We also experienced a similar issue. When RNA is isolated from stationary phase culture, the quality is poorer (according to RQN or RIN value) than RNA isolated from log phase culture. We used commercial kit RNeasy Plus Mini Kit from Qiagen. The obtained RIN values for all stationary phase RNA samples were above 7.5 which is acceptable for the high throughput sequencing, whereas the RIN/RQN values of log phase RNA samples were more than 8.5. We also observed that following purification of mRNA, incubation at room temperature leads to RNA degradation proportionally. I would like to encourage you to use commercial RNA isolation kit and must use DNase treatment following RNA isolation to increase yield and quality of RNA samples for high throughput experimentation.
Best wishes.
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