Back in graduate school, I purified a lot of RNA, and after a while, I became fairly successful at it. My yields were good, and the RNA was intact. However, many of my early attempts at RNA isolation yielded degraded RNA that did not work well in many downstream applications. In my case, successfully isolating high-quality RNA required practice. During my trials and tribulations, I learned a lot of tricks and tips about how to obtain high-quality RNA. Here I share some of these tricks to help you speed through that “practice makes perfect” phase so that you can isolate RNA like a pro.
First, a disclaimer: Even though I learned a lot during my time in the lab, I’m not saying that I was a pro when it came to RNA isolation. After months of “practice”, my RNA looked really good but never quite as good as that of one of the PostDocs in our lab, and I have to admit that I experienced an occasional twinge of jealousy. Even if you never reach RNA purification perfection, the information presented here should help you improve your RNA isolation results.
When isolating RNA, your biggest enemy is ribonuclease (RNase) activity. RNAses are everywhere: your hands, your benchtop, even dust particles in the air. They are also notoriously difficult to inactivate. Autoclaving reagents and equipment will inactivate some but not all RNase activity. The use of chelating agents also does not protect against all RNases because many of these enzymes do not require divalent cations for activity. One of your best lines of defense is an RNase-free environment. For more information about how to create an RNase-free environment, visit the RNA Isolation section of the Promega Protocols and Applications Guide.
Another big concern is proper sample collection and preparation. These initial steps can make or break your chances of obtaining high-quality RNA. The goal is to lyse the cells and inactivate RNases as quickly as possible. Snap freeze your samples and store them at –70°C if you can’t homogenize them immediately. Not only does this protect your RNA, but it also minimizes any unintentional changes in gene expression. This is particularly important for bacteria because bacterial RNA has a short half-life. To complicate matters, these cells often have complex cell walls that are not easily disrupted by standard chaotropic agents, so mechanical disruption and/or digestion with a lytic enzyme such as lysozyme is often necessary. When harvesting animal tissues, collect the sample as soon after dissection as possible. Be aware that some tissues such as pancreas and spleen have higher levels of RNases, so you will want to harvest these tissues before you harvest tissues that have lower levels of RNases such as liver. I always had good results freezing tissue samples in liquid nitrogen then breaking the tissue into smaller pieces in a mortar and pestle with additional liquid nitrogen before adding the lysis buffer. Be gentle and tap the tissue with the pestle; be too exuberant and you could end up with rat kidney in your hair. Be sure to let most of the liquid nitrogen evaporate before adding lysis buffer, or you’ll be trying to homogenize a block of ice. For woody plant tissues and other samples that contain high levels of polysaccharides, RNA purification protocols often include the surfactant cetyltrimethyl ammonium bromide (CTAB) to bind and remove the polysaccharides, which can interfere with downstream applications such as RT-PCR.
Homogenize the samples thoroughly in a lysis buffer that contains denaturants such as guanidine or urea to disrupt the cells and inactivate RNases. To aid cell lysis and homogenization, lysis buffers also may contain detergents in addition to denaturants, especially for samples with a high lipid content such as brain. Be sure that the samples stay cold. I recommend homogenizing samples on ice. After the initial lysis steps, many protocols incorporate an organic extraction to remove proteins and DNA. For maximal RNA purity, be sure to use the proper ratio of phenol:chloroform:isoamyl alcohol, and make sure that your reagents are of the proper pH so that DNA will partition into the organic phase, reducing DNA carryover into your RNA sample. Many protocols also incorporate a DNase digestion step to further reduce DNA contamination—just be sure that your DNase is RNase-free.
How do you determine if these tips and tricks improved your RNA purification prowess? Well, back in my day we ran an agarose gel with ethidium bromide and looked at the RNA to gauge RNA integrity. What I hoped to see was that the intensity of the 28S ribosomal RNA was about twice that of the 18S rRNA. After the first few months, I started to see the 45S rRNA consistently appear on my gels. Due to its size, the 45S rRNA was difficult to purify in one piece, making it a great marker for intact RNA. Nowadays, RNA gels are not as common as they once were. Instead, many researchers are using instruments such as the Agilent Bioanalyzer to measure RNA integrity.
Want more tips on how to obtain high-quality RNA and or just general tips about working with RNA? Visit these useful resources:
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