Anyone who has worked with RNA knows how challenging RNA can be and that there is much to consider compared to working with DNA. But why is RNA so challenging? How can you still obtain the best possible RNA sample? We want to summarize the most important points when working with RNA.
The first thing to realize is that RNA, unlike DNA, is not designed to be stable over time. This becomes clear when you consider the function of RNA molecules. Simply put, RNA is responsible for information transfer and control – not for information storage like DNA. RNA is single-stranded and contains ribose sugars instead of the double-stranded DNA’s deoxyribose sugars. Both structural differences make RNA unstable and more prone to degradation. Maybe the most challenging aspect of handling RNA samples is that the RNA-degrading enzymes, called ribonucleases (RNases), can be found almost everywhere. In the human body, RNases are used as a protection mechanism against invasive microorganisms. Thus, these enzymes are secreted in fluids such as tears, saliva, mucus, and perspiration. However, RNases can also be found, e.g., on flaky skin or hair. Moreover, RNases are very stable and resistant enzymes. Some do not even denature at temperatures of +100°C.
Therefore, there are a few aspects to consider when obtaining good-quality RNA samples. First and foremost, all measures must be taken to prevent RNase contamination of the working space during the isolation of RNA and further handling. In the following, additional relevant points are listed to bear in mind. Before starting RNA isolation, the surfaces of benches should be treated with commercially available RNase-inactivating agents. Additionally, the benches should be wiped with 100% ethanol to eliminate microorganisms. All glassware and plastic consumables that will be used must be RNase-free and sterile, and glass containers need to be treated with heat accordingly. It is necessary to always wear clean gloves and a lab coat. Objects that have not been treated with RNase inhibitors should not be touched. Also, treated objects should still be touched as little as possible. Touching the skin should be strictly avoided. Gloves should be changed frequently. Generally, a good sterile technique is necessary; tubes and bottles should be kept closed whenever possible, and coughing, sneezing, or breathing over open containers should be avoided. RNA-containing samples should be kept on ice and pipetted gently during all working steps.
After successfully isolating the RNA, it needs to be stored safely. Due to its general susceptibility to degradation, RNA should always be stored at -80°C. Even trace amounts of RNase can compromise RNA integrity. The storage at -80°C prevents the activation of possible RNase residues. The storage can be done in grade water, 10 mM Tris-HCl pH 8, or an elution buffer free of EDTA. Figure 1 shows the most important utensils for working with RNA. Keep them ready when starting your RNA project.
Finally, your samples are prepared and stored safely. They can now be shipped to our facility for sequencing. The samples should be kept on dry ice only to ensure they do not thaw during shipment. Once your samples arrive, we will take care of them to avoid contamination and degeneration at every process step.
Figure 1 | Most important utensils when working with RNA.