Ribbon diagram of RNA’s biggest threat: a ribonuclease
RNA analysis from RT-pPCR to RNA-seq has become an increasingly important part of life science research as we seek to understand gene expression patterns, cell signaling and developmental events. To be successful at these RNA analysis steps, however, the upstream RNA purification needs to produce intact, high-quality product suitable for downstream work. Many RNA purification systems are available, ranging from high-throughput to manual using a variety of chemistries. You can purify RNA from FFPE or fresh mammalian tissues. How do you know which system to choose and when to use it? Our free webinar on August 11, The Hows and Whys of Early Steps in RNA Analysis, describes different methods for purifying RNA from fresh or fixed samples, protecting it from degradation and assessing quality before you proceed with downstream work. Register today to learn how you can achieve the best results possible with your RNA analysis studies.
DNA and RNA Purification Product Selector
Working with RNA (blog)
About the Webinar Series
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“RNase A”. Licensed under CC BY-SA 2.5 via Wikimedia Commons – https://commons.wikimedia.org/wiki/File:RNase_A.png#/media/File:RNase_A.png
RNase, back in the early 1990s, posed a serious threat to laboratories working with RNA isolation. My graduate work involved isolating RNA from the tissues of Lyme disease-infected mice and hamsters. We struggled to DEPC-treat glass and plasticware, or autoclave anything that could be autoclaved, kept tissues cold during RNA harvest and held our breaths (truly, as aerosol could be another source of ribonuclease) until PAGE proved us successful in RNA isolation.
Ribonuclease (RNase) was omnipresent and the arch rival of our work, across several species, due to its RNA destroying abilities.
Now, a July 13, 2015 publication by researchers at the University of Wisconsin-Madison provided both a catch-up for this former lab rat on modern day research with and knowledge of RNase, as well as an exciting look at what may be a real purpose for this RNA-destroying molecule: RNase has moved to clinical trials due to the discovery of it’s cytotoxicity for cancer cells.
Raines’ group in the Department of Chemistry at UWI-Madison published in ACS Central Science their findings on the ligand that RNase 1 uses to attach to human cancer cells, in the article, “Human Cancer Antigen Globo H is a Cell-Surface Ligand for Human Ribonuclease 1”. Continue reading
Set up a lab RNA Zone
Working with RNA can be a tricky thing…it falls apart easily, and RNases (enzymes that degrade RNA) are ubiquitous. Successfully isolating RNA and maintaining its integrity is critical, especially when sensitive downstream applications are used (e.g., RNA-Seq).
Good techniques for RNA handling are simple to employ but crucial for success. All RNA purification and handling should take place in an RNase-free, RNA-only zone of the lab. Segregating RNA work from protein and DNA purification and handling will help minimize the potential for RNase contamination and help keep your RNA intact. Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab, and gloves should be worn at all times to prevent accidental contamination. Tools and equipment such as pipets, tips, and centrifuges should be designated for use only in the RNA zone as well. The location of the RNA zone in the lab is also important. Keeping traffic to a minimum and moving the RNA zone away from doors, windows, and vents can also help minimize contamination.
Using an RNase inhibitorcan also help safeguard your samples from RNase degradation. These inhibitors can bind to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present.
Water and buffer stocks can be a source of RNase contamination. Several stocks from an RNase-free zone in an academic lab showed RNase activity. Recombinant RNasin® inhibitor protected all RNA samples from degradation.