StudiesEvaluating Tools and Techniques for RNA Purification As a prerequisite for many molecular biology experiments involving real-time quantitative PCR, reverse transcription PCR, microarrays, Northern blot analysis, nuclease protection assays, RNA mapping, in vitro translation and cDNA library construction, RNA is an indispensable, though troublesome molecule. Due to its inherent susceptibility to ubiquitous RNases and its chemical instability, RNA is readily endangered by base- or enzyme-catalyzed hydrolysis. Researchers must take into account a variety of factors, which influence their ability to obtain high quality RNA that is free of contamination such as Rnases, proteins and genomic DNA. These factors include yield variations, processing requirements, and sample availability of different cells and tissues. Because of its mercurial nature, The Science Advisory Board wanted to examine the types of reagents and techniques scientists use in their isolation and purification protocols in an effort to identify best RNA purification practices. We questioned over 800 scientists about their methodology, applications, degree of satisfaction, and preferred supplier(s). A summary of their responses is presented below. The full list of responses can be accessed via the following link: http://scienceboard.net/studies/past/55comments.asp Typically, to avoid RNA degradation, efficient processing of tissue and cell samples for RNA isolation is critical. However, scientists typically experience problems when extracting RNA from different types of cells and tissues. To overcome this technical difficulty, slightly more than half of them use some means of mechanical cell/tissue disruption in RNA purification. The two most popular techniques are homogenization with a dounce and grinding/milling in liquid nitrogen. While mechanical disruption is the most common means of facilitating distraction, it is often a tedious procedure. The drudgery of this manual chore is a compelling reason for why almost 40% of scientists turn to enzymatic methods for cell/tissue disruption in RNA purification. The two most widespread techniques are GITC lysis solution and lysozyme followed by lysis buffer. “Finding a good method of homogenizing, particularly skin samples is also important because grinding skin in liquid nitrogen is a long process.” -Laboratory Tech, North America RNA is most often purified in order to perform reverse transcription PCR or quantitative real-time PCR. Scientists report that over a quarter of all their RNA samples are purified from established cell lines. There is an almost even split between the percentage of scientists who use commercially available acidic phenol/GITC reagents and those that use commercially available purification kits to purify their RNA. When respondents who use RNA purification kits were asked to indicate their primary supplier, they overwhelmingly chose Qiagen. Scientists chose Invitrogen and Sigma-Aldrich as their leading suppliers of commercially available phenol/GITC reagents. Scientists value kits that reproducibly produce high yield and high quality RNA. They also value ease-of-use and scalability. However, their expectations with respect to yield and throughput are not often fully realized. In addition to wanting all of the qualities they prize in their RNA purification kits, scientists also demand speed from their purification reagents. “I think that a superb RNA isolation kit should focus on three things: speed of preparation, protection from degradation during the whole procedure and low price.” -Principal Investigator, Central/South America Of the remaining 12% of scientists who do not use commercially available reagents or kits to purify RNA, homebrew methods prevail. According to study respondents, the most compelling reason to use these homebrew methods is price. “Kits are necessary, but not sufficient for intact and pure RNA. I found them too expensive, given that you can manufacture your own.” -Staff Scientist, Central/South America “However, price is not the only defining reason for the popularity of home-brew methods,” explains Tamara Zemlo, Ph.D., MPH, Executive Director of The Science Advisory Board. “An optimized protocol that results in both high yield and purity is an important consideration.” “I usually extract RNA from animal or human brain tissue. The use of kits like RNeasy (Qiagen) tends to give low yield of RNA due to the presence of brain lipids. Therefore, I use a two-step procedure. First, I purify the RNA with homebrew GdCN-reagent or TRIzol and in a second step apply this "crude" RNA to the RNeasy spin columns to obtain pure RNA with high yields.” -Staff Scientist, Europe Scientists with the highest level of throughput are much more likely to use robotic purification systems. While the number of these scientists is relatively small—5%, these researchers account for a large percentage of the total samples from which RNA is purified. Many scientists admit that automation would help them increase throughput, which is one of their unmet needs. However, most are hampered both by the initial cost of the equipment and the dearth of instrumentation that meets their lab’s exact specifications. “My environment is relatively conservative when it comes to automatization of RNA purification methods. Any robotic method or instrument must have a proven track record, with associated publications in peer-reviewed journals, in order to receive the blessing of our higher management at the time of purchase. This is probably because the process is parallel to our main expertise, as opposed to central or integrated with it. If robots were cheaper, the side-task of RNA purification could probably take sufficient importance to warrant a purchase. As it stands, because RNA purification and isolation are only tools that serve a higher purpose, any method that gets the work done will do.” -Lab Director, North America ### For insights into what life scientists view as the greatest challenge with regard to RNA purification, please click here. Further discussion occured in The SAB Forum, which can be accessed by clicking here. [ View Current & Future Studies ] [ View Past Studies ] |
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