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,
* Missouri Western State College, Saint Joseph, MO
64507; Pomona College, Claremont, CA 91711;
Genome Consortium for Active Teaching,
Davidson College, NC 28035
Submitted September 17, 2004; Revised December 6, 2004; Accepted December 15, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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Key Words: cDNA • gene expression • microarrays • mRNA • quantitative PCR • real-time PCR • undergraduate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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As instructors at a National Science Foundation-sponsored Genome Consortium for Active Teaching (GCAT) workshop at Georgetown University in July 2004, two of us (L.H. and T.E.) worked closely with a diverse group of 18 faculty from undergraduate institutions across the country (http://www.bio.davidson.edu/Biology/GCAT/workshop2.html). The participants learned to conduct microarray experiments and analyze microarray data. In the course of the workshop, the gel electrophoresis-based method used at Missouri Western State College for measuring complementary DNA (cDNA) levels was presented to the group and it generated a large amount of interest. We began to consider that the need to make this type of measurement without an automated real-time PCR instrument is widespread in undergraduate settings. A three-part plan was developed to address this issue. First, faculty at undergraduate institutions would be surveyed to gauge the need for a reliable and affordable alternative to real-time PCR and determine whether the level of interest in the workshop group is reflected in a larger group. Second, two of us (L.H. and L.C.) would conduct an experiment to evaluate the reliability of gel electrophoresisbased measurements made by undergraduate researchers in comparison to real-time data from the same samples. Third, dissemination to the undergraduate educational community would be accomplished in the form of this article.
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VALIDATING MICROARRAY DATA WITH REVERSE TRANSCRIPTION-PCR |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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SURVEY OF EDUCATORS |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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Of the responding faculty, 77% reported that they have reason to measure cDNA levels of individual genes. Examples of applications described in response to the survey include the measurement of changes in cDNA levels when bacteria are exposed to compounds that induce or repress heme precursors, the study of genes expressed during sexual development in a basidiomycete fungus, and the examination of the impact of changing chromosome copy number on levels of gene expression in Tetrahymena. Despite the widespread interest among the faculty in measuring individual mRNA levels, only three of the respondents have performed real-time PCR experiments, and only six have a real-time PCR machine at their institution. Six others have plans to purchase a real-time instrument once funds have been obtained through grants or institutional sources, and eight reported that they have a budget for real-time PCR reagents. However, all but one of the respondents have access to the thermal cycler and gel electrophoresis equipment needed to implement a gel electrophoresis-based quantitative PCR method. Of the responding faculty, 80% replied that they would find a gel electrophoresis-based method for quantifying mRNA useful in a laboratory course and 87% reported that they would be able to use it in an undergraduate research setting. These results reinforced our initial observation from the GCAT workshop that the need for a convenient, inexpensive, and reliable alternative to real-time PCR is widespread at undergraduate institutions.
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OPTIONS FOR MEASURING mRNA LEVELS |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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, where C is the
difference in the number of cycles required to yield the same amount of
product in the two reactions. These considerations led to reports within the past 15 yr of several different methods for the measurement of cDNA levels by quantitative PCR (Bustin, 2002; Halford et al., 1999). The methods vary widely with respect to the cost of the required equipment and reagents and also as to the ease with which they can be adapted for use in undergraduate settings. The most popular method of late is automated real-time PCR (Bijwaard et al., 2001; Gibson et al., 1996; Heid et al., 1996). A real-time PCR instrument is usually a combination of a thermal cycler and a fluorimeter that enables the investigator to monitor the products of a reaction while it is proceeding. The ability to measure DNA sensitively and quantitatively allows the determination of the point in a reaction when the amount of the amplification product of cDNA exceeds a user-defined threshold. With proper controls, the cycle at threshold (Ct) gives a measure of the initial cDNA level. The least expensive and most straightforward way of measuring product is through the use of a fluorescent dye specific for double-stranded DNA, such as SYBR Green I. After each cycle of PCR, the reaction mixture is exposed to light at the absorption wavelength for the dye, and emission is used as a measure of the amount of product. However, this method detects not only the specific product, but also products resulting from nonspecific priming and primer-dimer formation. Therefore, optimization of reactions is critical, and real-time PCR machines use melting profiles after a reaction has been conducted to determine how much of the product is specific. The issue of specificity can be bypassed with more expensive gene-specific probe systems. Molecular beacons contain a reporter fluorophore and a quencher on their 5' and 3' ends that produce fluorescence only when the central part of the oligonucleotide is bound through specific base pairing to the PCR product to be measured (Abravaya et al., 2003). TaqMan probes (http://www.appliedbiosystems.com) bind specifically to the cDNA template in an area central to the region being amplified, and emit fluorescence only after a quencher is removed from the 5' end by the action of Taq polymerase (Pongers-Willemse et al., 1998). Real-time PCR is a sensitive and accurate method, but it involves expensive equipment and reagents.
Before the advent of real-time PCR, the literature described several gel electrophoresis-based PCR methods developed to measure mRNA levels. Each of these uses reverse transcription to produce cDNA and quantitative PCR to measure the level of cDNA for selected genes. One such method, competitive reverse transcription-PCR, controls for RNA stability and differences in the efficiency of reverse transcription (Gilliland et al., 1990; Vu et al., 2000). Control RNA of a different size than the mRNA to be measured is produced by in vitro transcription of the coding sequence cloned into the appropriate plasmid. The control RNA is added in known amounts to the RNA sample and cDNA is produced. PCR with primers that amplify cDNA templates from both the control RNA and the specific mRNA is then conducted. Gel electrophoresis of the products and densitometry of band intensities give a measure of the absolute level of mRNA for the selected gene in the original RNA sample by calibration against the known standards. Competitive reverse transcription-PCR is a sensitive and accurate method, but it requires time-consuming procedures for cloning and transcribing a control RNA for each gene to be studied.
Another gel electrophoresis-based method, primer dropping, employs amplification within the same reaction of cDNA from both the gene to be measured and a control gene (Wall and Edwards, 2002). If the mRNA from either of these two genes is present at substantially higher levels than the other, one cDNA amplification product accumulates at levels past the exponential phase of the reaction before the other reaches the threshold of detection. Primer dropping allows one of the two amplifications to proceed for a small number of cycles before primers for the other amplification are added, ensuring that both genes are in the exponential phase of the reaction at the same time. In this way, band intensities can be used to measure the mRNA levels of a gene when they are substantially different from the mRNA levels for the control gene. This method did not meet our need for a flexible and convenient method that could be easily and quickly applied to any gene of interest from a microarray experiment.
We chose to adapt a method of quantitative amplification of cDNA that is based on the exponential relationship between the amount of accumulated product and the initial template level (Noonan et al., 1990). Identical amplifications are conducted with primers for a given gene and cDNA template derived from two RNA populations to be compared. Before the inflection point of the curve of product versus cycle number, the ratio of product amounts for the two reactions is proportional to the ratio of initial cDNA templates present. The amount of product present at several points in the exponential phase of the reactions can be measured by densitometry of the intensities of bands after electrophoresis. In this way, the relative amounts of cDNA in two samples can be measured. Before making measurements with this method, it is important to consider several sources of experimental error. Because RNA samples can vary in quality, and the efficiencies of reverse transcription and PCR are not constant, Noonan et al. (1990) employed an internal control. Primers for the amplification of the cDNA for a ubiquitously expressed gene were included in the reaction along with primers for cDNA from the gene to be measured. This approach lacks adaptability because it is difficult to optimize the annealing temperature for a duplex reaction, and unwanted products can appear. An alternative approach is to use parallel PCR amplifications to measure the cDNA levels from an experimental gene relative to those of a reference gene (Lesur and Campbell, 2004). Because this method is better suited to undergraduate settings, we chose to develop it in our research laboratory and to compare it with microarray analysis and real-time PCR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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Measuring RNA Quantity and Quality
Elimination of contaminating yeast genomic DNA in RNA preparations is
crucial before cDNA synthesis and PCR. We tested each RNA sample by amplifying
with primers for the housekeeping gene TDH1. Although the RNA
isolation procedure includes a DNase I digestion, about half of our samples
showed a product, indicating DNA contamination. We therefore performed a
second DNase I treatment with twice the enzyme recommended by the RNA
isolation kit and for twice as long, then performed the PCR check again.
Spectrophotometric readings at 260 and 280 nm were then made for the RNA
samples. The yield of RNA varied from 20 to 30 µg from each 5-ml yeast
culture and the A260-A280 ratios were between 1.8 and
2.1. As a further quality check, 1 µg of each RNA sample was subjected to
denaturing agarose gel electrophoresis. The presence of 28S and 18S ribosomal
RNA over a background smear of mRNA and the absence of both degraded RNA and
high molecular weight DNA indicated good quality RNA preparations.
Microarray Procedure
Total RNA from DAPI-treated and untreated yeast was used as starting
material for the Genisphere 3DNA Array 900 Expression Array Detection Kit
(http://www.genisphere.com/).
The RNA was reverse transcribed with Superscript II (Invitrogen Life
Technologies,
http://www.invitrogen.com)
using primers that include a capture sequence for fluorescent dendrimers.
Hybridization of the cDNA to an Institute for Systems Biology 70 mer
oligonucleotide DNA chip provided by GCAT was performed with 40 µl of
hybridization buffer (0.5 M NaPO4, 1% SDS, 2 mM ethylenediamine tetraacetic
acid, 23 saline-sodium citrate, and 43 Denhardt) at 588C overnight. The 3DNA
Cy3 and Cy5 fluorescent dendrimers were hybridized in hybridization buffer for
4 h at 588C. Dithiothreitol (DTT) at a level of 1 mM was added to the wash
solutions. The chip was scanned at Davidson College by Dr. Malcolm Campbell.
We used Scanalyze
(http://rana.lbl.gov/index.htm?software/)
to grid the microarray data and collect intensities from the two channels.
Reverse Transcription
Reverse transcription of total RNA for the preparation of cDNA to be used
in PCR was also performed using Superscript II as the reverse transcriptase,
but with oligo(dT)15 primers. A solution of 5 µl RNA (0.2
µg/µl), 1 µl deoxynucleotide triphosphate (dNTP) mix (10 mM each),
and 5 µl dH2O was heated to 658C for 5 min and then chilled on
ice. To this mixture was added 4 µl of 53 first-strand buffer, 2 µl of
0.1 M DTT, and 1 µl RNasin Plus RNase inhibitor. The contents were gently
mixed, briefly centrifuged, and allowed to incubate for 2 min at 428C. Reverse
transcriptase was added (1 µl), and the reaction was incubated for 50 min
at 428C. The reaction was stopped by heating at 708C for 15 min.
Polymerase Chain Reaction
Polymerase chain reaction was performed in 40-µl reactions containing 84
pmol of each of two primers, 4 µl of 10 mM dNTP mix, 4 µl of Mg-free 103
buffer (100 mM Tris pH 9.0 at 258C, 500 mM KCl, 1% Triton X-100), 4 µl 25
mM MgCl2, and 2 units Taq DNA Polymerase. The PCR reagents were
assembled as a master mix that was split into reactions that received 1 µl
of cDNA from either treated or untreated cells as template. PCR was performed
with cycles of 948C for 15 s, 588C to 628C for 15 s, and 748C for 30 s.
Aliquots of 7 µl of the reactions were removed after a determined number of
cycles and two-cycle intervals thereafter until four samples were taken from
each 40-µl reaction. We designed primers for the amplification of cDNA from
each of three yeast genes. The forward primer
5'-ATGTCTGAATCAGTGGCCATTATAGGTGC-3' and reverse primer
5'-ATCAATGCCACGAGCAGAAATAGCC-3' yielded a 180-base pair (bp)
product from BNA4 using an annealing temperature of 628C. For OLI1, a 124-bp
product was produced by the forward primer
5'-GAGCAGGTATGGTATTGCTATCGT-3' and the reverse primer
5'-CCTGTAGCTTCTGATAAGGCGAAACC-3', with annealing at 588C. Forward
primer 5'-ATCGATGTCGCTGTTGACTCCACT-3' and reverse primer
5'-CAATGGAGCCAAACAGTTGGTGGT-3' gave a 206-bp product for
TDH1 at an annealing temperature of 608C. Although the TDH1
reverse primer is also predicted to bind to the paralogs TDH2 and
TDH3, the forward primer is specific for TDH1.
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Real-Time PCR
An ABI Prism 7000 was used for real-time PCR analysis of the three selected
genes. Amplification reactions were assembled as described above but with ABI
23 SYBR Green I Master Mix comprising half the total volume, and with the
total volume per reaction adjusted to 50 µl. The cDNA templates were
diluted two-, four- and eight-fold, and three replicate reactions were
performed for each.
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RESULTS AND DATA ANALYSIS |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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Using DAPI as a model for minor groove binding compounds, we cultured yeast in its presence and absence. We then isolated RNA from the two cultures for use in microarray analysis. Figure 1 shows the microarray results from the three selected genes. The cDNA derived from untreated yeast was labeled green, and that from DAPI-treated yeast was labeled red. The spot for TDH1 is yellow, with a ratio of treated to untreated of 1.1, indicating that its mRNA is present at equal levels in the two samples. The spot for BNA4 is red, with a treated to untreated ratio of 4.5, indicating a higher level of mRNA from DAPI-treated cells. OLI1 mRNA levels are lower in the DAPI-treated sample, because it is green, with an untreated to treated ratio of 8.3. These results are indicated in Table 2. In addition, the spot intensities give an indication of the relative abundance of mRNA species. The mRNA for BNA4 appears to be present at a lower level than that for the other two genes.
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We subjected the very same RNA samples used for microarray analysis to the gel electrophoresis-based method for measuring cDNA levels. Accordingly, mRNA from DAPI-treated and untreated yeast cultures was converted into cDNA and subjected to PCR with primers for the three selected genes. The polyacrylamide gel electrophoresis of reaction samples taken at two-cycle intervals from 16 to 22 cycles for TDH1 and OLI1 and from 20 to 26 cycles for BNA4 is shown in Figure 2. The images show that the specific PCR product for TDH1 accumulates at an equal rate in the treated and untreated cDNA reactions. For BNA4, the product appears much earlier in the treated reaction, while the opposite is true for OLI1. The intensities of these bands were measured by using a digital camera and the densitometry feature of Kodak Digital Science 1D version 2.0.3. The values were used to calculate relative intensities compared with the 100-bp band of the molecular weight marker on each gel.
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The cDNA preparations from the DAPI-treated and untreated RNA isolations were subjected by L.C. and L.H. to real-time PCR analysis using the same primers and PCR conditions as for the gel electrophoresis-based method. An ABI Prism 7000 was used to determine the fluorescence of DNA-bound SYBR Green I as a function of the number of cycles. Because SYBR Green I measures the total double-stranded DNA that accumulates in the reaction tube, it is important to determine how much of the PCR product is specific and how much arises from nonspecific priming and primer dimer formation. For this reason, each reaction was subjected to a dissociation protocol after the cycles were complete. The expected result of a well-optimized reaction is a product that melts at a single, reproducible melting temperature characteristic of that DNA sequence. Other PCR products will usually display lower melting temperatures. For the three genes selected, the dissociation protocols indicated the production of only minor amounts of products that melted at temperatures 10-20 degrees below the main peak, in agreement with the gel results above. However, for BNA4, the dissociation peak was broader, covering almost 10 degrees, while more typical product melts, including those for the other two genes in this study, cover 2-3 degrees. This observation is in accord with the appearance of a slight array of background bands on the electrophoresis gel loaded with BNA4 PCR products. We therefore decided to check whether the BNA4 product from the gel electrophoresis experiments was specific by digesting it with four different restriction endonucleases. The 180-bp product was cut at position 164 by HaeIII, position 105 by MwoI, position 111 by TaqI, and positions 135 and 165 by Tsp509I, as predicted by the DNA sequence. These results verify the identity of the specific BNA4 product that was quantified by the gel electrophoresis method. In this case, measuring a specific band with the gel electrophoresis-based method gives a cleaner result than SYBR Green I real-time PCR, because only the correctly sized products are quantified.
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There is a good relative correspondence between the threshold numbers of cycles required for detection with the gel electrophoresis-based and real-time PCR methods. The data indicate that BNA4 mRNA levels are higher in the treated than in the untreated sample, while the opposite is true for OLI1. The larger number of cycles for BNA4 product detection indicates the lower level of its cDNA, as was shown with the microarray analysis.
Real-time Ct values were also used to produce ratios of initial cDNA
levels. Ct values from the three replicates of each dilution of cDNA template
were averaged and the differences, DCt, between reactions with treated and
untreated cDNA, were calculated. Considering that the accumulation of product
is given by P = (1 + E)C, where P is the amount of product, E is
the efficiency of amplification, and C is the number of cycles, the plots of
fluorescence versus number of cycles were used to determine the efficiency for
each of the reactions. The efficiencies varied only slightly, from 80% to 82%,
and were used with the DCt values in the formula I1/I2 =
(1 + E)Ct to calculate ratios of treated to untreated
initial cDNA levels. The relative levels of initial cDNA present in untreated
versus treated samples are reported in
Table 2. The standard
deviations reflect variation between measurements made with three dilutions of
the template. The ratios of treated to untreated mRNA levels are 1-8 for
TDH1 and 6-4 for BNA4, and the ratio of untreated to treated
mRNA levels is 9-8 for OLI1.
We used an independent samples t-test to compare the mean ratios reported in Table 2 for the gel electrophoresis-based and real-time methods. For the induced gene BNA4, the two methods produced the same conclusion because there was no statistically significant difference between the means (p = .24). Similarly, means for the repressed gene OLI1 were not statistically different (p = .66). However, the analysis did reveal a difference between the mean ratios for TDH1 (p = .012). This may mean that that the real-time method is more sensitive, or that proprietary components in the SYBR Green I Master Mix provide improved consistency between reactions. The measurement of a slight induction of TDH1 as a result of our experimental treatment indicates that it may not be the ideal choice of a housekeeping reference gene under all conditions. However, the difference in TDH1 results from the microarray and gel electrophoresis-based methods to real-time PCR does not disallow the conclusion that BNA4 is induced and OLI1 is repressed. Although this analysis reveals differences in the results from the two methods of quantifying individual mRNA levels, they are both in good general accord with the microarray results. The encouraging conclusion with regard to the development of a reliable gel electrophoresis-based method is that all three methods revealed the same trends for the effects of treatment on the three genes studied. The treatment increased the steady state mRNA level for BNA4, decreased it for OLI1, and had little effect on TDH1. Considered as a whole, the results support the conclusion that the gel electrophoresis-based method is a convenient and economical way to reliably quantify steady state mRNA levels.
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EDUCATIONAL EFFECT ON UNDERGRADUATE RESEARCHERS |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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The students were also asked to comment on the overall objective of this study to develop a method that is student friendly and appropriate for use in undergraduate teaching and research settings. Sara said, "it was not very difficult to learn how to use the method, and I had only minor questions throughout the procedure. I think this will provide a tremendous amount of learning opportunities for undergraduate students." According to Dan, "the method is appropriate for use in undergraduate teaching laboratories, and its implementation into undergraduate curricula will reinforce the central dogma of molecular biology. The ease with which the method can be performed and the fact that it was developed by undergraduates illustrate its fitness for undergraduate teaching and research laboratories."
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QUANTIFYING mRNA LEVELS IN UNDERGRADUATE LABORATORIES |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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RECOMMENDATIONS |
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TOPABSTRACTINTRODUCTIONVALIDATING MICROARRAY DATA WITH...SURVEY OF EDUCATORSOPTIONS FOR MEASURING mRNA...MATERIALS AND METHODSRESULTS AND DATA ANALYSISEDUCATIONAL EFFECT ON...QUANTIFYING mRNA LEVELS IN...RECOMMENDATIONSREFERENCES |
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We recommend that care be taken when choosing a housekeeping gene for use as a reference. This point is illustrated by the fact that although the TDH1 gene we used as a reference appeared to be unaffected as measured by microarray analysis and the gel electrophoresis-based method, the real-time PCR data showed a slight effect of the treatment. A better choice may be the yeast gene TUB1, because its transcript was found to be stable and unregulated in a series of experiments involving aging in yeast cells (Lesur and Campbell, 2004). Although a one-sample t-test showed expression ratios for TDH1 from 18 microarray experiments from our laboratories to be statistically different from unity (p < .01), the same analysis showed TUB1 expressed to be unchanged (p = .39).
We would also like to point out some alternatives to the procedures we used. There are several choices available for RNA isolation kits. Because the isolation of good-quality RNA is key, we suggest looking at the Ambion Web site for "Ten Ways to Improve Your RNA Isolation" (http://www.ambion.com/techlib/tn/91/9113.html). The negative control experiment of using RNA samples in an amplification to test for contaminating DNA could be omitted if the primers span an intron. In this case, genomic DNA would reveal a larger size product in the amplification of cDNA. Our selected genes, and 95% of yeast genes in general, do not contain introns. PCR products could be measured using agarose gel electrophoresis instead of polyacrylamide gel electrophoresis. Although we have found that polyacrylamide gels produce tighter bands that are more easily measured with densitometry, agarose gels may be easier to use in a teaching laboratory setting. If needed, a fluorophore that detects smaller amounts of DNA, such as SYBR Green I, could be used in place of ethidium bromide to stain gels. If a digital camera and software system designed for gel analysis are unavailable, densitometry could be performed using a normal digital camera and a software package such as NIH Image (http://rsb.info.nih.gov/nih-image/) or ImageJ (http://rsb.info.nih.gov/ij/).
We also recommend that time be invested in the optimization of reactions before an attempt to use either real-time PCR or the gel electrophoresis-based method is made. This process begins with primer design; we used the PrimerQuest tool at Integrated DNA Technologies (http://scitools.idtdna.com/Primerquest/). We used an annealing temperature of 4 degrees below the Tm reported by the manufacturer and conducted four reactions with cDNA template and MgCl2 concentrations varying from 1.5 mM to 4.5 mM. Using the MgCl2 concentration that yielded the fewest nonspecific products and least primer-dimer, we then optimized the annealing temperature. We suggest using "hot start" PCR as a next step, if needed (Santoyo et al. 1997). Although it is possible to exclude from measurement nonspecific products that migrate differently during gel electrophoresis than the specific product, we urge caution in this approach. It is also possible that the nonspecific and specific products have the same mobility. The use of a reaction that is not completely optimized can also be problematic in real-time PCR employing SYBR Green I, as opposed to a gene-specific probe system. We recommend that the specific product be verified by restriction digestion whenever possible.
A final important consideration is that even though the gel electrophoresis-based method agreed favorably with microarray and real-time data for three selected genes, it is not the most reliable method available. That real-time PCR holds this position is well accepted. We recommend that the gel electrophoresis-based method be used as a convenient and inexpensive alternative in undergraduate teaching and research settings where real-time PCR is impractical. It could also be used in a transitional role between microarrays and real-time measurements. For example, microarray experiments could be used in an undergraduate research project to generate a list of genes, and the gel electrophoresis-based method could be used to determine which of these measurements are the most reliable. Data of this sort would be suitable for local or even national presentations, or publication in an undergraduate journal. The mRNA levels for selected genes should then be further validated by real-time PCR before data are used in a professional publication.
The results of this study demonstrate that the gel electrophoresis-based method can produce data for quantifying mRNA levels that are in accord with microarray analysis and real-time PCR. The results validate the use of the method in our undergraduate research group at Missouri Western State College, allowing us to address important questions in an economical, yet reliable way. The dissemination of the method also provides opportunities for the surveyed faculty and many others to engage their undergraduate students, tomorrow's scientists and teachers, in technology that is fundamental to the new genomics era.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address correspondence to: Todd T. Eckdahl (eckdahl{at}mwsc.edu).
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