Methods and reagents: Is your fluorometer on the blink? Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses some common problems associated with measuring DNA concentrations using a mini-fluorometer and the handling of RNA samples. For details on how to partake in the newsgroup, see the accompanying box. That blinking fluorometer! ************************** Netters have known for some time that large discrepancies are found between DNA concentrations determined from spectrophotometric readings and those from a fluorometer, with differences in the DNA concentrations being as high as 1000-fold. The difficulty might stem from inconsistency in baseline measurements, which netters say tends to drift, making sample readings unreliable. Others blamed the difference on the more common problem of contaminating RNA, which could throw off any calculated DNA concentration, already discussed in this column [TiBS 19, 93-94 (1994)]. However, low purity of DNA cannot explain why so many netters have encountered the same problem with the same machine: the Hoefer TKO 100 mini-fluorometer, which, although no longer made by Hoefer, is still used in many labs. Some think that it is often a gamble to use the TKO 100 because they claim there is some kind of variation between individual units - some work while others do not. They say the machine is frustratingly difficult to use and it does not perform any better, even though Hoefer offered a `fix' a couple of years ago. Some netters think the reasons for the differences in performance could be due to sensitivity to traces of organic chemicals or detergents, interference from dust or condensation, misalignment of the light beam, or that the internal electronics are prone to fluxes within the power supply or proximity to other electronic equipment. While the problem of dust can easily be dealt with by dispensing reagents from a closed container with dust-free pipette tips, chemical interference might be more difficult to overcome because the contaminating substance could be coming from solutions used for the DNA extraction (see reply from Hoefer on next page). Philip L. Carl (plc@med.unc.edu) showed that the components of a standard mini-prep kit can misrepresent the amount of DNA in the samples. When a mock alkaline lysis mini-prep was performed without bacterial cells, the resulting DNA-free supernatant assayed by fluorometry gave false readings indicating the presence of DNA in the sample. Therefore, samples derived this way might be causing the problem. Not everyone is unhappy with the TKO, however, and many netters do not understand why other people have problems. One person wrote that the fluorometer is very sensitive as long as no ethidium bromide is present in the sample because ethidium bromide will compete with the Hoechst dye and give a gross underestimate of the actual fluorescence. In addition, DNA isolated from agarose gels using affinity columns or bound to glass particles might not be completely devoid of ethidium bromide. Those who have used the machine for years say that while the fluorometer might not provide absolutely accurate values of DNA concentration, it provides consistency within the same lab. When compared with the standard method of eyeballing DNA samples on an ethidium bromide stained agarose gel, the fluorometer should win hands down. RNase contamination: fact or fiction? ************************************* When working with RNA, many people give great attention to keeping their solutions free from RNase and they take great pains to avoid losing their precious RNA samples to nucleases, which could cause havoc and possibly disrupt weeks or months worth of experiments. Clean RNA handling has traditionally been done with utmost care including the wearing of long-sleeve lab coats and latex gloves to cover exposed arms and hands which are thought to be sources of skin-borne nucleases. Some people even go as far as wearing hair nets in order to rid their work area of any possible contaminating RNases coming from fallen hairs. Being fearful of the dreaded unseen RNase, RNA workers typically sterilize all their equipment, bake and autoclave all their glassware, use autoclaved tips, and treat their water supply with diethylpyrocarbonate (DEPC). However, netters have varying opinions about the amount of care with which one must treat RNA samples and about the source of contaminating RNases. Many are not convinced that such a great deal of effort put forth to kill RNases is necessary and that the omnipresence of RNases in the laboratory is nothing more than a myth. One common fallacy is that autoclaving solutions will destroy RNases, but netters say that autoclaving plastic tips and glassware will not kill the RNases. They say that these practices are most likely done out of laboratory ritual. Some feel that being careful to remove endogenous RNases and not to introduce new RNases into an RNA prep is much more important than worrying about nucleases that may have already made their way into premade solutions, mainly because nucleases will most likely not be found in distilled water, properly cleaned glassware, new plastic, or on most people's fingers. Another experienced RNA worker wrote that DEPC has a much greater potential for inhibiting subsequent enzymatic manipulations than it does for protecting RNA. DEPC inactivates RNases mainly through modification of the imidazol ring of histidine residues, but it must be removed by heat treatment afterwards. There have been at least two reports of DEPC treatment interfering with enzyme reactions posted to the `methods' group. Netters now suggest that water and other solutions be tested for RNase first, and if they test negative, the solutions should not be treated with this dangerous chemical. Rimantas Plaipa (Rimantas.Plaipa@GF.VU.LT) of Vilnius University, Lithuania, found that the efficiency of DEPC treatment and autoclaving for destroying RNase activity strongly depends on pH and on the composition of the solution. Interestingly, he found that DEPC is destroyed within Tris, MES, and Hepes buffers within minutes and so it is unsuitable for treatment of common molecular biology buffers anyway. He warns that one cannot be sure that DEPC inactivates RNases under all circumstances and that it might not inactivate nucleases irreversibly. Therefore, current practices may be insufficient in eliminating RNases from these solutions. Skin RNases? ************ Opinions also differ about where the nucleases come from. Some netters think that only certain people extrude RNases from their hands, while others do not shed them. As a test for the presence of nucleases on his hand, one netter described doing an RNase assay on himself by tipping a microcentrifuge tube of solution against his finger tip, shaking the tube, and pipetting 10 ul into a tube containing 2 ug of RNA markers. After the tube was incubated for 30 minutes at 37 degrees C, no RNase activity was observed as the standards appeared the same when run on a gel. However, another netter ran a similar test for comparison against the activity of bovine pancreatic RNase. After applying 20 ul of buffer solution to the tip of his finger and waiting for a minute, the activity of RNases in the solution was approximately the same as 10 ng ml-1 of pancreatic RNase, revealing the presence of a similar nuclease. In one of the original papers published on skin RNase, the authors washed two whole fingers from six individuals in 10 ml of solution and obtained similar activities of 1-10 ng ml-1 of pancreatic RNase for each. [1] Self-splicing RNA ***************** Rimantas Plaipa also posted a very interesting study on `the possible involvement of RNases in the so called "RNA self-splitting" at Y-A bonds' which was recently presented as a poster in the first meeting of the RNA Society in Madison, WI, USA, and is soon to be published. While performing RNA processing experiments, small amounts of products corresponding to cleavages of CA or UA frequently appear, with the extent of cleavage varying from virtually none seen to complete digestion of the RNA, which could ruin any RNA work. Rimantas wrote that while working on a project involving self-splicing RNA, he arrived at the conclusion that many so-called intrinsic RNA self-cleavages by ribozymes are probably artifactually caused by contaminating RNases. As both skin RNase and pancreatic RNase are highly specific for UA and CA sequences, they produce the same banding pattern during PAGE as claimed for many self-cleaving RNAs. He says that experiments show RNase inhibitors completely suppress some previously published self-splicing reactions. He thinks that mature tRNAs are evolutionary optimized to be very resistant to RNases under native conditions, but that pre-tRNAs are much more sensitive. If treated carelessly, these precursors might appear to have the self-cleavage ability. He also opposes the hypothesis concerning differences between people with respect to hand nucleases because he has done experiments that suggest skin RNases are quite widespread if not universal in the human population. In addition, the distribution of the RNase activity found throughout the laboratory suggests that, much like the artifactual banding seen on SDS-PAGE gels [see TiBS 21,191-193 (1996)], the contamination originates from the settling of dead human skin cells rather than from direct touching of tubes or equipment with the workers' bare hands. As the RNase activity is thought to be ubiquitous, how does one go about eliminating it? There are three main ways to rid your work area: wash plastic and metal instruments with an alkaline solution, treat buffers and solutions with DEPC or filter them, and then autoclave everything. Netters suggest that for any RNA work, buffers should be made fresh from powders, then frozen and replaced often and at critical times. Frozen aliquots should be replaced entirely if any problems do arise. When combined with a rigorous benchtop washing with a solution of 100 mM NaOH, many routine RNA rituals can be eliminated and with them the threat of the unseen nucleases. Reference ********* [1] Holley, R. W., Apgar, J. and Merrill, S. H. (1961) J. Biol. Chem. 236,PC42-PC43 ******************************************************************************* Reply from Hoefer Pharmacia Biotech, Inc. Over the years, customer's comments, suggestions and requests have been considered and incorporated into a redesigned, microcontroller-based fluorometer called the DQ 200. This new model has many new features that makes it both more reliable and simple to use than the TKO 100, and every effort has been made to ensure that line voltage fluctuations do not affect readings in any way. Application notes are provided to illustrate the pitfalls and nuances of fluorescence-based DNA quantiatation, including PCR product quantitation and plasmid DNA calibration. SEAN GALLAGHER Technical Director Hoefer Pharmacia Biotech, Inc. 654 Minnesota Street San Francisco, CA 94107, USA. ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/aug96.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1996. Methods and reagents: Is your fluorometer on the blink? Trends in Biochemical Sciences 21(8):317-318. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************