Analysis of the RNA content of the yeast Saccharomyces cerevisiae.
These limitations are unfortunate in light of the growing body of scientific information about the pre-biotic RNA world. It now seems clear that the basic steps in protein synthesis were established before DNA became important as a way of storing genetic information in a stable way (Gesteland et al., 2006; Gilbert, 1986; Muller, 2006; Spirin, 2002; Woese, 2001). In addition to having self-catalytic activity, RNA molecules are involved in all the steps of translation, from the initial activation of amino acids by attaching them to transfer RNAs to the association of messenger RNAs and aminoacyltransfer RNAs with ribosomes to the actual polymerization of amino acids into polypeptide chains. Table 1 summarizes the characteristics and sizes of the major types of RNA commonly found in prokaryotic and eukaryotic cells. The Howard Hughes Medical Institute (2006) has produced a DVD from its Holliday Lectures on Science series which discusses the many roles of RNA and can be used to introduce students to this topic. In this article, we describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. The general goals of these experiments are to emphasize the importance of RNA in cell biology and to provide practice in basic biochemical and molecular analysis.
Overview of Experiments
This set of experiments focuses on RNAs from the yeast Saccharomyces cerevisiae, a unicellular budding microorganism that has served as a model of cellular and molecular processes in eukaryotes (Davis, 2003). S. cerevisiae can be grown easily in the laboratory, has a relatively small genome that has been completely sequenced, and is susceptible to genetic analysis using both classical and molecular techniques. In these experiments, students:
1. study yeast cells by light microscopy
2. estimate the number of cells in a liquid culture using three different methods
3. extract RNAs from yeast cells for biochemical analysis
4. prepare a RNA standard curve using the orcinol reagent
5. determine the concentration of RNA in their extract
6. calculate the total amount of RNA per cell
7. prepare a mini-prep of highly-purified yeast RNA suitable for molecular analysis
8. separate the RNA molecules in the mini-prep by horizontal gel electrophoresis
9. determine the sizes of the major RNA species that are present in their sample.
Figure 1 shows a flow chart of these experiments as we have used them in a sophomore-level Cell Biology course. In the next sections, we describe the individual experiments and give examples of the data that can be obtained. In some cases, several variations are suggested so that instructors can adapt the experiments to their particular situation. We then discuss some alternative ways of scheduling these studies and several pedagogical and technical issues that have arisen in using these experiments in our course. At the end of the article, we list the key solutions needed for the experiments.
Microscopic Observations of Yeast Cells
We provide students with 25 ml of a liquid culture of yeast cells that have been grown aerobically at 30[degrees]C in yeast extract/peptone/dextrose (YPD) medium overnight. While we normally use a haploid laboratory strain (SEY6210) that has been used in genetic studies, one can also use commercial samples of Fleischmann's[R] or Red Star[R] dry active yeast from the grocery store. We have the students begin the experiment by examining the cells microscopically so that they can see what these organisms actually look like. The yeast cells can be easily seen in simple wet mounts of the liquid culture using either bright-field or phase-contrast optics (Figure 2). Like other industrial yeasts, the two commercial samples are aneuploids (Codon et al., 1998) and visibly larger in size than SEY6210. All three samples typically show a large number of budding cells.
Counting of Yeast Cells
In order to quantify the amount of RNA per yeast cell, it is necessary to determine the number of yeast cells per ml of the liquid culture. This can be done in three ways. First, students can measure the turbidity or optical density of the culture in a spectrophotometer at a wavelength such as 600 nm. Because the overnight cultures are usually quite dense, it is necessary to make a 1/10 dilution in YPD medium first. While the turbidity value does not directly indicate the number of cells per ml, it does correlate with cell concentration and can be used as an estimator of it. Second, students can use a hemocytometer to count the cells microscopically. Again, it is helpful to use a 1/10 dilution of the overnight culture so that there is a reasonable number of cells to see on the slide. We usually have students score five of the large squares on a standard hemocytometer, and then average the counts before calculating the total number of cells per ml. Finally, students can estimate the number of cells in the liquid culture by doing a viable cell or plate count. They can make serial 1/10 dilutions of the overnight culture in YPD medium and spread 100 [micro]l portions of the dilutions on YPD agar plates with a sterile glass, metal, or plastic spreader. We usually make the dilutions in YPD medium using micropipettors and sterile microcentrifuge tubes (100 [micro]l + 900 [micro]l in a 1.5 ml tube), but larger volumes, sterile glass (or plastic) pipets, and sterile glass test tubes could be used if necessary. The plates are incubated at 30[degrees]C for three days and then refrigerated prior to scoring. The 10-5 and 10-6 dilutions usually give the most accurate colony counts and are used to calculate the number of viable cells per ml of culture. The accuracy of the viable count can be improved by having the students make duplicate plates of these dilutions. Typical results from two separate experiments are shown in Table 2. Because the commercial yeast strains (Fleischmann's[R] or Red Star[R] dry active yeast) are larger in size, they usually give higher turbidity values but lower total or viable cell counts per ml.
[FIGURE 1 OMITTED]
Chemical Extraction of RNAs & Other Macromolecules
To determine the total amount of RNA per cell, it is necessary to extract the RNAs and other macromolecules from the yeast cells. We have students prepare an extract that is suitable for biochemical analysis using an abbreviated version of a procedure previously described (Rendina, 1971; Deutch & Parry, 1974). The cells in a 10 ml portion of the yeast culture are harvested by centrifugation for five minutes at about 3000 rpm in a clinical centrifuge in a 15 ml conical plastic centrifuge tube and washed with 5 ml of 0.85% NaCl. The cells then are disrupted by suspending them in 5 ml of ice-cold 10% trichloroacetic acid (TCA) for 20 minutes. This extracts the small molecules from the cells, but precipitates the nucleic acids, proteins, and carbohydrates. After another five-minute centrifugation, the pellet of insoluble macromolecules is washed with 5 ml of 95% ethanol, and the nucleic acids are extracted with 5 ml of 5% perchloric acid (PCA) for 20 minutes at 70 [degrees]C. After a final five-minute centrifugation, the supernatant fraction containing the RNAs in 5% PCA is decanted and saved for analysis. The pellet containing proteins and carbohydrates is discarded.
Preparation of an RNA Standard Curve
The RNA content of the 5% PCA extract can be determined using the orcinol reaction (Schneider, 1957). In this procedure, solutions containing RNA are heated in the presence of HCl, orcinol, and ferric chloride. Heating in the presence of a strong acid causes depurination (the release of the purine bases) and hydrolysis of the RNA strands to give free pyrimidine nucleotides, ribose, and phosphate. The ribose undergoes dehydration to form furfural, which reacts with orcinol and ferric chloride to form a green-colored product. To quantitatively measure the RNA, a standard curve is first prepared using a 100 [micro]g/ml stock solution of RNA from baker's yeast. Varying amounts of the stock solution and water are combined in 13 X 100 mm glass tubes to give a total sample volume of 1.0 ml. Freshly-prepared orcinol reagent (3.0 ml) is then added to each tube using a repipettor, and the solutions are heated at 100[degrees]C for 15 minutes in a heat block. After cooling the tubes, the absorbance of each solution is read at 660 nm in a spectrophotometer. The resulting standard curve usually shows good linearity (Figure 3). The slope of the line can be used to convert any unknown absorbance within the range of the standards to a particular amount of RNA in [micro]g.
Determination of Yeast RNA Concentrations
To determine the total amount of RNA in the 5% PCA extract of yeast cells, varying volumes (100, 200, 300, or 500 [micro]l) of the extract and 1/10 or 1/100 dilutions of it in 5% PCA are combined with water to give a total volume of 1.0 ml. It is necessary for the students to test different volumes and dilutions of the PCA extract because the amount of RNA in it is unknown. Orcinol reagent (3.0 ml) is again added to each tube and the samples are heated at 100[degrees]C for 15 minutes. After cooling, the absorbance of each solution is determined at 660 nm. The absorbance values of those samples within the range of the standard curve are used to determine the RNA concentration of the PCA extract in mg of RNA per ml. For example, if 300 [micro]l (0.3 ml) of a 1/10 dilution of the PCA extract gives an absorbance at 660 nm of 0.235, the RNA concentration based on the standard curve shown in Figure 3 can be calculated as:
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[FIGURE 2 OMITTED]
Our students usually find that several tubes give absorbance values within the range of the standard curve, and so do this calculation for each individual sample. They then determine the average for the PCA extract as shown by the sample data in Table 2.
Calculation of RNA Content per Yeast Cell
From the value of the RNA content of the 5% PCA extract in mg per ml and the total cell count per ml value obtained with the hemocytometer counts, the RNA content of the yeast cells can be calculated (Table 2). It is necessary to take into account that 10 ml of the yeast culture were used to prepare 5 ml of the 5% PCA extract. The calculation for the first sample in the Table (SEY6210, Experiment 1) is therefore:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]
As expected, the RNA content is higher for the larger aneuploid strains. Boehlke and Friesen (1975) reported that the total RNA content of the haploid yeast strain A364A was about 0.4 pg per cell after growth in a low nutrient medium called yeast nitrogen base. Yeast cells grown in an enriched medium like YPD are considerably larger and so have a higher RNA content.
Isolation of Highly Purified Yeast RNA
To isolate high-quality RNAs from the yeast cells that are suitable for gel electrophoresis, we have students prepare a "mini-prep" using the YeaStar[TM] kit of reagents and a small spin column (Zymo Research Corporation). This method is much simpler than more traditional chemical extractions and avoids the use of toxic chemicals like phenol and chloroform. A 1.5 ml sample of the yeast culture is transferred to a 1.5 ml microcentrifuge tube and centrifuged at 10,000 rpm for one minute in a high-speed microcentrifuge. The medium is removed and the cells are suspended in 80 [micro]l of Digestion Buffer. Zymolase[TM] enzyme (5 [micro]l) is added and the sample is incubated at 37[degrees]C for 60 minutes. Lysis buffer (160 [micro]l) is added and the suspension is centrifuged at 10,000 rpm for two minutes. The liquid lysate is removed with a micropipettor and added to a small spin column provided with the kit, which is then centrifuged for one minute at 10,000 rpm to bind the RNAs to the supporting resin. After two washes with 200 [micro]l portions of Wash Buffer, the RNAs are eluted with 60 [micro]l of RNAase-free water into a sterile microcentrifuge tube.
Agarose Gel Electrophoresis of RNAs
The RNAs in the mini-prep sample can then be analyzed by horizontal gel electrophoresis to identify the predominant types of yeast RNA. We have the students do this in 1.2% agarose gels with a simple tris-borate-EDTA (TBE) nondenaturing buffer. While RNAs are sometimes analyzed under denaturing conditions in gels containing formaldehyde or DMSO/glyoxal (Ausubel et al., 2002), we prefer to use this method because it avoids the use of toxic reagents like formaldehyde and does not require buffer recirculation during electrophoresis. Different volumes of the mini-prep RNAs and a "ladder" of RNA size markers (New England BioLabs) are combined with the 2X RNA sample buffer that is provided with the ladder to give a total sample volume of 10 [micro]l. The 2X sample buffer contains 7 M urea to help denature the RNA. The samples are heated at 65[degrees]C for three minutes, cooled, and loaded in the wells of the gel. The RNA samples are run at 150 volts for 45 to 50 minutes in TBE buffer. The gels are then removed, stained with a dilute solution (about 0.5 [micro]g/ml) of ethidium bromide for 15 minutes, and washed with water for 15 minutes. The resulting bands are then visualized with a UV-transilluminator (Figure 4). Two intense bands of RNA are normally visible, along with several other fainter bands.
[FIGURE 3 OMITTED]
Determination of the Sizes of the Major Yeast RNAs
In order to identify the major RNAs recovered from the yeast mini-prep, it is necessary to determine their size. The distances migrated by the RNA fragments in the "ladder" are measured and used to construct a mobility standard curve in which the log10 of the fragment size is plotted as a function of the distance migrated (Figure 5). From this standard curve, the sizes of the RNAs in the yeast mini-prep can be estimated. The two major species of RNA seen in the gels have sizes of about 2200 and 3900 nucleotides, respectively. Similar RNAs are seen in all three samples of yeast. These RNAs correspond to the two largest ribosomal RNAs: the 18S rRNA from the smaller subunit (1789 nucleotides; Rubtsov et al., 1980) and the 25S rRNA from the larger subunit (3392 nucleotides; Georgiev et al., 1981). The difference between the published sizes and those measured here reflects the fact that while the RNAs are denatured by heating in a sample buffer containing urea prior to loading, the gels are run in a nondenaturing TBE buffer. Ribosomal RNAs are known to have extensive secondary structure due to intrachain hydrogen bonding (Gutell et al., 2002), and incomplete denaturation of the RNAs will cause the molecules to move more slowly through the gel. The RNAs thus appear to be larger than they actually are. Additional RNA bands are also visible in some of the preparations, which probably correspond to precursors or intermediates in ribosomal RNA formation. While yeast RNA preparations are commercially available which are suitable for use as standards in the orcinol assay, they are too degraded for the electrophoretic analysis (Figure 6A). Although ethidium bromide gives the greatest sensitivity in terms of staining the RNA bands after electrophoresis, it is possible to stain the gels with a less toxic methylene blue-based reagent. However, the intensity of the bands after staining with this dye is much less and so the bands are harder to see unless higher concentrations or larger volumes of RNA are used (Figure 6B).
We like this series of experiments because it is quantitative with respect to the RNA analysis and because it introduces students to agarose gel electrophoresis in a different context than is usually the case. The experiments are interdisciplinary in the sense that they involve elements of biology (microscopy and cell structure, RNA and protein synthesis), chemistry (acidic extraction of macromolecules, formation of colored products from RNA by chemical reactions), physics (absorption of light by molecules in a spectrophotometer, movement of RNA molecules in an electric field), and mathematics (construction of standard curves, sequential numerical calculations). They thus provide a good opportunity for students to tie various topics in their curriculum together.
[FIGURE 4 OMITTED]
The experiments can be scheduled in many different ways depending on the particular interests of the instructor and the availability of supplies and equipment. We usually do these experiments as part of a two-week project in a sophomore-level Cell Biology course. The students in this course have one three-hour lab session each week and are organized into lab groups of three to four students. Prior to this project, the students to this project, the students do a series of simpler one-week experiments in which they learn how to use micropipettors and to make serial dilutions, how to use the Spectronic 20 Genesys spectrophotometers we have for class use, and how to construct and properly use standard curves. In the first week of this project, they carry out the microscopic observations of the yeast cells, make the various cell count determinations, and prepare both the 5% PCA extract and the RNA mini-prep. The extract and the RNA mini-prep are then stored in a -20[degrees]C freezer until the next lab session. In the second week of the project, they count the colonies from the viable count determination, do the orcinol assays for RNA content, and perform the agarose gel electrophoresis. We use small Owl B1A gel systems and visualize the bands with a Bio-Rad VersaDoc digital imaging system, but any similar apparatus or UV transilluminator will work. The lab sessions are busy, but the students usually get everything done on time. For those instructors who have shorter lab sessions or who want to spread the work out over more lab periods, it would be feasible to freeze the yeast culture before doing the chemical extraction, to spend full periods on construction of the RNA standard curve and the analysis of the PCA extract, and to spend additional periods on the RNA mini-preps and the agarose gel electrophoresis. For those instructors who want only to do the agarose gel electrophoresis experiment, it would be possible to give the students a small yeast culture and have them make the RNA mini-prep in one lab session, and then run the agarose gel in another lab session.
[FIGURE 5 OMITTED]
There are many variations on these experiments that instructors might want to consider:
1. Students could compare the RNA contents of different yeasts as shown here.
2. Students could analyze the RNA content of the same yeast after growth in either an enriched medium like YPD or a more minimal medium like yeast nitrogen base.
3. Students could study the RNA content of the same yeast in the active exponential phase of the growth cycle or in the stationary phase of the growth cycle.
4. Students could compare the RNA content of haploid and diploid forms of the same yeast.
Technical Problems, Preparation of Materials & Laboratory Safety
The project raises a number of important technical issues for the students to consider. First, counting the yeast cells themselves is not a trivial matter, in part because the cells grow by budding. Large buds often appear as separate but neighboring cells in the hemocytometer counts, but even a cell with a large bud will form a single colony in a plate count. In comparing the viable counts to the total counts, it may appear as though only a fraction of the cells are actually alive. This is particularly a problem with strain SEY6210, which is a haploid strain with smaller cells. Because of the buds, the calculated values of RNA content per cell are probably an overestimate of the true values. Second, the agarose gel electrophoresis requires that students analyze their gels carefully. The mobility of the RNA fragments, like the mobility of DNA fragments or the mobility of proteins in SDS-PAGE gels, is a function of the log10 of the molecular size rather than the size itself. There are two ways to graph the data. Students can calculate the log10 of the size of each fragment and plot these values as a function of distance as shown in Figure 5. Alternatively, they can use semi-log graph paper and plot the fragment size directly as a function of distance. This type of standard curve is new for many students and so requires some careful discussion.
[FIGURE 6 OMITTED]
The various solutions required for this experiment are summarized in Table 3. Most are relatively simple to prepare. The YeaStar[TM] RNA kit costs about $100, but it contains enough of each reagent for 40 preparations. We have used a single kit for several semesters in a row. The trichloroacetic and perchloric acids used in the chemical reaction are strong acids and need to be handled carefully. The orcinol reagent is made up in concentrated HCl and should be treated as hazardous. We have occasionally had problems with the reagent, due to using the wrong container of ferric chloride. It is important to use Fe[Cl.sub.3].6 [H.sub.2]O rather than an anhydrous or ferrous form of the salt. We use a repipettor to dispense the orcinol reagent into the tubes in each assay and cover the tubes with aluminum foil during the heating step to avoid HCl vapors. Some instructors may want to handle the reagent themselves and do the dispensing and heating steps in a fume hood. The RNA ladder used in the agarose gels costs about $60 for 50 [micro]l of solution at a concentration of 500 [micro]g/ml (0.5 [micro]g/[micro]l). The solution can be stored at -70[degrees]C for several semesters. A 2 [micro]l portion (1 [micro]g) is sufficient to obtain clearly visible bands if ethidium bromide staining is used, but a larger portion would be better if the methylene blue stain is employed. The ethidium bromide solution is potentially mutagenic and must be treated carefully. As indicated in Figure 6, a methylene blue-based stain could be used as an alternative. The yeasts themselves, however, are not pathogenic, and can be handled using basic microbiological techniques.
Evaluation of Student Understanding
In our Cell Biology course, we use several different assessment techniques to evaluate student understanding of these experiments. First, each lab session begins with a short 15-minute laboratory quiz consisting of 10 multiple-choice questions. About half of the questions are designed to test understanding of the previous week's lab work and about half are designed to assess preparation for the new work. Some of the questions are numerical and are used to see if the students have understood how to do the relevant calculations. Second, each lab group turns in a "data sheet" summarizing a particular week's lab work at the beginning of the next lab session. The data sheet includes places for students to enter the actual data collected as well as to present images of gels, standard curves, and numerical calculations. Third, this set of experiments forms the basis of the first full-length lab report for the semester, which we ask students to write in the standard scientific format using the book by McMillan (2006) as a reference. The fact that there are several different parts to the project forces the students to keep track of the various methods and to connect several different pieces of data together. The complexity of the project also illustrates the value of dividing both the Materials & Methods section and the Results section of the lab report into smaller subsections. The set of experiments is challenging for many students. It requires good teamwork, and while some groups of students try to divide up the work on the data sheets and lab reports, full understanding of the experiments depends on every student's participation. These experiments also involve the use of basic mathematical skills outside of a math classroom, which emphasizes the importance of thinking about why a particular calculation is done in a certain way.
This set of experiments introduces students to RNA in a way that can be successful in a teaching laboratory for undergraduates or even for high-school students. Because we often tend to focus on messenger RNA as a key intermediate linking DNA and protein in lectures about cellular and molecular biology, students are sometimes surprised to find that the predominant species of RNA in their mini-prep are the larger ribosomal RNAs. However, it is the high concentration of ribosomes in yeast cells that allows their rapid rate of reproduction and the efficient translation of messenger RNAs. The experiments thus highlight the importance of ribosomes and the contribution they make to the translation process.
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CHARLES E. DEUTCH (email@example.com) is Associate Professor, and PAMELA A. MARSHALL is Assistant Professor, both in the Division of Mathematical and Natural Sciences, Arizona State University at the West Campus, Phoenix, AZ 85069.
Table 1. Properties of the major types of RNA molecules in cells. Number and Size Range (nt = nucleotides, Type of RNA Description S = Svedberg units) mRNA Encodes the amino acid Varies based upon gene messenger RNA sequence for size, but ranges from polypeptide synthesis 400-10,000 nt rRNA Necessary for In prokaryotes, there are ribosomal RNA ribosomal function; three rRNA molecules with ribosomes are made of approximate sizes of 120 two subunits nt (5S), 1400 nt (16S), constructed of both and 2900 nt (23S) polypeptides and rRNA molecules In eukaryotes, there are four rRNA molecules with approximate sizes of 120 nt (5S), 160 nt (5.8S), 1850 nt (18S), and 4600 nt (28S) In Saccharomyces cerevisiae there are four rRNA with sizes of 121 nt (5S), 158 nt (5.8S), 1789 nt (18S), and 3392 nt (25S) Mitochondria and chloroplasts also have their own rRNA sequences that vary in size depending on species tRNA Covalently bind amino There are 20 different transfer RNA acids for delivery to types of tRNA molecules ribosomes for protein (one for each amino acid) synthesis; the with approximate sizes of codon-anticodon 70-90 nt (4S) interaction is critical for translation to occur 7S RNA Only in eukaryotes; 300-400 nt (7S) required as a part of SRP (signal recognition particle) for targeting of ribosomes to the endoplasmic reticulum (ER) for transport of polypeptides into the ER scRNA Only in prokaryotes; 250-400 nt (4.5S) small cytoplasmic required for protein RNA secretion; function is similar to 7S in eukaryotes RNA subunit of The enzyme RNase P is 350-410 nt RNAase P necessary for processing of bacterial tRNAs to their functional length; the RNA molecule is the catalytic subunit and the protein subunit acts as a cofactor and helps tRNA binding snRNA Components of the There are five major small nuclear RNA eukaryotic splicosome, species of snRNA, termed which is necessary for U1, U2, U4, U5, and U6, splicing and removal approximately 100-600 nt of introns in the premRNA molecules snoRNA Components of the There are 77 genes small nucleolar nucleolus in encoding snoRNA in S. RNA eukaryotes; required cerevisiae, approximately for processing of rRNA 100-600 nt molecules to mature functional rRNA Viral RNA genomes Some viruses contain Variable based upon RNA rather than DNA species of virus 3000-1 for their genome million nt siRNA Complementary Varies based upon mRNA small interfering interfering RNAs that that is targeted, usually RNA can lead to mRNA 20-25 nt inactivation Based on a table from Brooker (2005) with information for S. cerevisiae from the Saccharomyces Genome Database (www.yeastgenome.org). Table 2. Summary of RNA analysis in various yeast strains. Total Count Strain Exp [OD.sub.600] (yeasts/ml) SEY6210 1 4.15 1.76 x [10.sup.8] 2 4.95 2.24 x [10.sup.8] Fleischmann's[R] 1 5.65 1.24 x [10.sup.8] 2 7.06 1.91 x [10.sup.8] Red Star[R] 1 6.03 1.61 x [10.sup.8] 2 7.45 2.51 x [10.sup.8] Viable count RNA in extract Strain Exp (yeasts/ml) (mg/ml) SEY6210 1 2.19 x [10.sup.8] 1.01 2 1.56 x [10.sup.8] 1.08 Fleischmann's[R] 1 1.20 x [10.sup.8] 1.70 2 1.52 x [10.sup.8] 1.86 Red Star[R] 1 1.24 x [10.sup.8] 1.75 2 2.14 x [10.sup.8] 1.77 RNA/cell Average Strain Exp (pg/yeast) (pg/yeast) SEY6210 1 2.87 2.64 2 2.41 Fleischmann's[R] 1 6.85 5.86 2 4.87 Red Star[R] 1 5.43 4.48 2 3.52 Table 3. Preparation of solutions and materials. For growth of yeast cultures and cell counts: liquid YPD medium: 20 g peptone, 10 g yeast extract, 20 g D-glucose (dextrose), 1000 ml water; autoclave to sterilize YPD agar plates: 20 g peptone, 10 g yeast extract, 20 g D-glucose (dextrose), 15 g agar, 1000 ml water; autoclave to sterilize and pour into sterile plastic petri dishes For extraction of RNA from yeast cells: 0.85% NaCl: 8.5 g NaCl, 1000 ml water; autoclave to sterilize 10% TCA: dilute concentrated 100% liquid trichloroacetic acid 1/10 with water 5% PCA: dilute concentrated 70% perchloric acid 1/14 with water For orcinol assay of RNA: RNA standard: 100 [micro]g/ml RNA from baker's yeast (sigma-Aldrich R6750, $80.20/100 mg) in water orcinol reagent: add 0.5 g Fe[Cl.sub.3].6 [H.sub.2]0 to 500 ml of concentrated HCl and mix carefully; add 15 ml of a solution of 1.5 g orcinol (Sigma-Aldrich 01875, $18.80/5 g) in 95% ethanol; mix carefully; make up on day of the experiment and dispense with a repipettor. For isolation of RNA: YeaStar[TM] RNA mini-prep kit (Zymo research corporation R1001, $104.00/kit) For agarose gel electrophoresis: 10X TBE solution: 108 g Tris base, 55 g boric acid, 40 ml 0.5 M [Na.sub.2] EDTA, pH 8.0; dissolve in water and make up to 1000 ml 1X TBE buffer: dilute 10X solution 1/10 with water RNA ladder: 500 [micro]g/ml (New England BioLabs N0362S, $58.00/25 [micro]g, supplied with 2X RNA sample buffer) ethidium bromide: 0.5 [micro]g/ml in water (Sigma-Aldrich E1510, $42.40 for 10 ml of 10 mg/ml stock solution)
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|Title Annotation:||INQUIRY & INVESTIGATION|
|Author:||Deutch, Charles E.; Marshall, Pamela A.|
|Publication:||The American Biology Teacher|
|Date:||Nov 1, 2008|
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