The Data Collection Bit

To survive, each cell ha to express thousands of proteins to react to different stresses. These proteins are code for through RNA. When researchers want to know what a cell is up to, they look at a snapshot of the RNA in the cell. Though today was my first attempt to isolate the RNA from cells in my new lab, it will definitely not be my last.

The first step of the RNA analysis involved lysing the cells to release RNA. This was accomplished by exposing the cells to a buffer with a different pH than their usual, inducing apoptosis.

We then put the buffer and solution through a filter to remove the genomic DNA. The filter works by utilizing the differences in the DNA and RNA bases. DNA has the four bases adenine (A), thymine (T), cytosine (C), guanine (G) while RNA had adenine (A), cytosine (C), guanine (G), and uracil (U). The silica membrane filter “holds” onto the thymine, effectively filtering genomic DNA out of the solution.

The filter holding the DNA is discarded and the solution is run through a new filter, that “holds” onto most of the biomolecules in the solution including RNA. To remove other molecules that can interfere with the RNA analysis, we use the RW1 and RPE buffers. RW1 is a strong washing buffer that removes carbohydrates, proteins, fats acids, and other biomolecules, while RPE is a mild buffer that mainly removes salts. These buffers are used because having other biomolecules in the solution when trying to analyze it (or turn it into cDNA as we will do later) can disrupt the process.

At this point, we hopefully have a fair bit of clean RNA in our filters. To separate the RNA from the rest of the pellet, we add RNase free water. The water has to be RNase free so it won’t affect/destroy the nucleic acid in the filters. The water changes the pH of the filter, which releases the filter’s “hold” on the RNA, allowing to come to the bottom of the tube.

Though we think we have RNA now, the lab always wants to double check. To do so, we use the Nanodrop test. Nucleic acid absorb UV light in various wavelengths, so the percent absorption of UV light shining through an RNA solution can be used to determine how much RNA the sample contains. The Nanodrop test uses very small quantities (about a single microliter) of RNA solution to test for the amount of RNA. Using surface tension, it suspends the drop between two pedestals, and analyzes the absorbance of light through that drop, giving a measurement of the amount of RNA per microliter. If we determine that there is enough RNA (more than 4 nanograms per microliter), we move on to the next step.

RNA is unstable, so to perform a better analysis of which proteins are being expressed in the cells, the scientists prefer to analyze cDNA. cDNA can be created through reverse transcription of the RNA that we now have. The process is called reverse transcription because usually DNA is transcribed into RNA, but in this case it is the other way around.

To create analyzable amounts of cDNA we use polymerase chain reactions (also called PCR). PCR works by making a small quantity of cDNA using the reverse transcriptase, then allowing that DNA to replicate exponentially. Manual PCR of the cDNA would take far too long, so the lab uses a thermal cycler that switches between a “hot” and “normal” phase. In the “hot” phase, the hydrogen bonds between the bases of the two strands of DNA are broken. Each strand of DNA then undergoes replication to make its complimentary strand, that join together in the “normal” phase. The newly synthesized cDNA is then separated again through the “hot” phase. This cycle repeats hundreds of times.

Before all this, the first step of cDNA creation is to prepare an environment where cDNA can be created in even amounts. We always want to start with 300 microliter of RNA, so we calculate the amount of the RNA solution we need to add to get that amount of RNA with the equation “amount of solution needed = 300/(nanograms/microliter)”. However, we also need the solution volume to be equal, so if the amount of RNA solution needed is less that 10 microliters, we add more RNase free water to make up the difference. If the amount of RNA solution needed is less than 10 microliters, it is capped at 10 microliters. The PCR will also need materials to create cDNA, which is gets through MasterMix.