Week 3: All Roads Lead To RNA
Hi Everyone. Thanks for coming back to my blog! This week I extracted and isolated RNA from my pet angelfish’s tail fin tissue. In this post, I’ll discuss the science behind RNA extraction and then my experience with the process!
The science behind RNA extraction and isolation:
Extracting RNA from a cell is pretty easy. Most of it is found in the cytoplasm, a gelatinous liquid that fills the entirety of the interior of a cell. To extract the RNA, all you need to do is break open the cell membrane that surrounds the cytoplasm.
However, the hard part is RNA isolation. In other words, separating the RNA from everything else that’s found in the cell. From DNA and proteins to organelles and lipids, cells are a busy place. But for any downstream applications (like sequencing), RNA needs to be pure, with minimal contamination of these other cellular components. Molecular biologists have developed an innovative strategy to solve this challenge. Here’s how we broadly obtain and maintain pure RNA .
- Homogenize (break down) the animal tissue into smaller pieces.
- Lyse (break open the cell membrane) the cells that make up the tissue with a detergent.
- Add a proteinase enzyme to break down the cellular proteins.
- Add a DNAse enzyme to degrade DNA molecules.
- Perform a series of “washing” steps to further remove impurities like salts and phenols.
- Store the RNA in RNase free water or a buffer solution to ensure stability.
- Measure the concentration of RNA in a spectrophotometer.
- Store the RNA at -80°C to prevent degradation.
All of these steps help ensure that the purest form of RNA is obtained. Below is a photo of my lab bench with all the chemicals and equipment I needed to obtain high quality RNA.
Furthermore, RNA unlike DNA is notoriously unstable and degrades in mere hours at room temperature. Keeping the RNA at ultra low temperatures (Step 8) ensures the stability of the RNA and limits the ability of endogenous RNAses to degrade the RNA.
After following Steps 1-6 from above, I was ready to test whether I successfully extracted RNA from the angelfish tissue. To do this, I placed a small sample of my RNA on a spectrophotometer (Step 7). Here’s a picture of me doing just that.
Spectrophotometers work by shining different wavelengths of light through the loaded sample. Depending on how much light passes through, it can be calculated how pure your extracted RNA is and how much RNA you have. Below is the spectrophotometry data I obtained.
To summarize everything, I obtained approximately 3 µg (3 x 10-6 grams) of RNA from the angelfish tissue. This may not sound like much, but just know that a single cell has at most 30 pg (3 x 10-12 grams) or RNA! Furthermore, I only needed 200 ng ( 2 x10-7 grams) to perform sequencing. I obtained an order of magnitude more RNA than I needed! This will hopefully leave some room for error.
To determine the purity of the RNA samples, we look at the 260/280 and 260/230 wavelength ratios. For a pure RNA sample, the 260/280 ratio falls between 1.8 and 2 and the 260/230 ratio falls between 2 and 2.2. The ratios of my extracted RNA were 1.92 and 2.11 respectively which indicates that I was successfuly able to extract relatively pure rRNA with minimal contamination. Extraction and isolation was successful!
I had a lot of fun doing this work and I hope you learned a bit about nucleic acid purification in this post. I hope to see you again next week!