ARTICLE |
Correspondence to: Elizabeth R. Unger, Centers for Disease Control and Prevention, 1600 Clifton Road, MSG18, Atlanta, GA 30333. E-mail: eru0@cdc/gov
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Summary |
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We have optimized conditions for the chemiluminescent analysis of gene expression using high-density filter arrays (HDFAs). High sensitivity and specificity were achieved by optimizing cDNA probe synthesis, hybridization, and detection parameters. The chemiluminescent expression profile reflected expected differences in the transcripts isolated from different sources (placenta and keratinocytes). We estimated the detection limit for low-abundance message to be 115 transcripts per cell, a sensitivity rivaling that reported for microarray formats and exceeding that reported for autoradiographic HDFAs. The method allows for short exposure times and reuse of probe. It should be equally applicable to techniques such as differential screening of cDNA libraries and differential display PCR. (J Histochem Cytochem 47:337342, 1999)
Key Words: gene expression monitoring, chemiluminescent detection, high-density filter arrays, cDNA synthesis
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Introduction |
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Gene expression analysis has been revolutionized by the development of high throughput methods that allow simultaneous monitoring of ever-increasing numbers of genes through hybridization to arrayed targets (
We were interested in using HDFAs to screen for differences in the gene expression of peripheral blood lymphocytes from patients with chronic idiopathic fatiguing illnesses and controls. We anticipate that this screening approach will yield insight into the pathogenesis of these illnesses and provide novel markers for diagnosis and therapy. Because of the limited amount of each sample and concerns about hazardous radioactive waste, we optimized a nonradioactive labeling and detection system for HDFAs that would minimize hazards to personnel, permit short exposure times, and allow reuse of labeled sample. This approach is feasible only if the hybridization results accurately reflect differences in the mRNA population of the original samples and allow detection of low-abundance messages. Here we report optimized conditions for the synthesis and use of digoxigenin-labeled cDNA probes with sensitive and specific chemiluminescent detection of the gene expression profile. The ability of this approach to detect low-abundance message in a complex mixture of RNA transcripts was evaluated using serial dilutions of a known human RNA target as template in the cDNA preparation. We demonstrated that this approach will detect transcripts that comprise 0.001% of the total mRNA population.
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Materials and Methods |
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Poly(A)+ RNA Extraction
Human placental poly(A)+ RNA was purchased from Clontech Laboratories (Palo Alto, CA). Total RNA from a monolayer culture of neonatal foreskin keratinocytes (NFKc) was extracted by the modified guanidinium thiocyanate method (
Synthesis of DigoxigenindUTP Labeled cDNA Probes
Probes were synthesized in 20-µl reactions containing 1 µg poly(A)+ RNA, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 10 mM DTT, 0.5 µg oligo(dT)12-18, 100 ng random hexamers, 0.5 mM each dATP, dGTP, and dCTP, 0.13 mM dTTP, 0.07 mM dig-11dUTP (Boehringer Mannheim; Indianapolis, IN), and 200 U SuperScript II RT (Life Technologies; Gaithersburg, MD). Poly(A)+ RNA, oligo(dT), and random hexamers were mixed, heated to 70C for 10 min, and left on ice for the addition of all reaction components except the enzyme. This mixture was incubated at room temperature (RT) for 5 min, the enzyme was added, and the reverse transcription was allowed to proceed for 55 min at 42C. The reaction was terminated by heat inactivation at 70C for 15 min. The probes were treated with 2 U of RNase H for 20 min at 37C, heat-denatured at 95C for 3 min, and cooled on ice before use.
Evaluation of Digoxigenin-labeled cDNA Probes
Probe synthesis was evaluated by denaturing polyacrylamide gel electrophoresis. Two µl of a 1:10 dilution of the cDNA reaction was mixed with 8 µl of loading dye (95% formamide, 0.5 mM EDTA, 0.025% SDS, xylene cyanol, bromophenol blue) and electrophoresed through an 8 M urea/5% polyacrylamide gel at 150 V for 90 min using a Mighty Small II SE 250 electrophoresis unit (Hoefer Scientific Instruments; San Francisco, CA). After electrophoresis, dig-labeled cDNA was transferred to a Nytran Plus membrane (Schleicher & Schuell; Keene, NH) by electroblotting at 400 mA for 90 min with the Panther Semi-Dry Electroblotter (Owl Scientific; Woburn, MA). The digoxigenin-labeled cDNA was immobilized to the membranes by UV crosslinking and was detected by the chemiluminescence procedure described below.
Hybridization using Digoxigenin-labeled Probes
Hybridization of all nylon membranes was done using Dig Easy Hyb (Boehringer Mannheim). Hybridization of nylon membranes in ExpressHyb solution (Clontech), the solution recommended by Clontech for hybridization with radioactive probes, resulted in unacceptable background (not shown). Hybridizations were done in roller bottles. Membranes were prehybridized for 24 hr at 42C with 0.2 ml hybridization solution/cm2. Hybridization proceeded overnight at 42C with 0.078 ml hybridization solution/cm2. After overnight hybridization, the membranes were washed twice with 2 x SSC/0.1% SDS at 42C for 15 min each and twice with 0.5 x SSC/0.1% SDS at 68C for 30 min each.
Chemiluminescence Detection
Before detection, the membranes were blocked with 5% blocking solution (Boehringer Mannheim) for 2 hr at 37C. Digoxigenin was detected by incubation with a 1:10,000 dilution of anti-digoxigenin/alkaline phosphatase conjugate (Boehringer Mannheim) in 1% blocking solution for 15 min at 37C. Membranes were washed and incubated with CPD-Star (Boehringer Mannheim) as recommended by the manufacturer. The membrane was sealed and exposed to Lumi Film (Boehringer Mannheim) for various times.
Sensitivity of Expression Analysis
Phage promoters were added to a partial (422-BP) human leukophysin cDNA cloned into the pCR-TRP vector (GenHunter; Nashville, TN) using the riboprobe primer set and PCR conditions suggested by GenHunter. (The human leukophysin gene cloned into the pCR-TRP vector was received as a gift from M. Rajeevan, Atlanta, GA).
RNA transcripts were prepared from the PCR template using SP6 RNA polymerase and the MEGAscript in vitro transcription kit (Ambion; Austin, TX) according to the manufacturer's specifications. The quality and quantity of in vitro transcribed human leukophysin mRNA was measured by UV absorbance and gel electrophoresis with known amounts of a marker RNA. Various amounts of this human leukophysin mRNA (2200 pg) were spiked into 1 µg of Drosophila melanogaster poly (A)+ RNA from embryo (Clontech) to achieve dilutions (w/w) of 1:5000, 1:20,000, 1:50,000, 1:100,000, and 1:500,000. One µg of each dilution as well as Drosophila without leukophysin was used to synthesize digoxigenin cDNA as described above. The entire probe was used as described above in the hybridization and chemiluminescent detection of nylon membrane dot-blots prepared to simulate HDFAs. The dot-blots contained human leukophysin cDNA and various negative control DNAs (pUC19, X174 RF I, bacteriophage
, pBR322, M13mp18 RF I and human papilloma virus 16 L1 gene), each at 10 ng/spot, the same concentration used in the Atlas HDFA. The blots were prepared on positively charged nylon membranes (Boehringer Mannheim) by spotting heat-denatured DNAs followed by UV crosslinking and baking (75C for 1 hr).
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Results |
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Synthesis of Digoxigenin-labeled cDNA
Gene expression analysis using HDFAs or microarrays requires that the probe reflect the complexity of mRNA in the original sample. For methods using labeled cDNA prepared from poly (A)+ RNA, isolation of quality total RNA, poly (A)+ RNA purification, and labeling of cDNA must be reproducible and effective. Key variables in the cDNA labeling reaction include the reverse transcriptase (RT) enzyme, type of primer, nucleotide concentration, and fraction of labeled nucleotide, as well as the temperature of the reaction. We evaluated each of these variables in turn, using 1 µg of human placental poly(A)+ RNA as the standard template. The size and extent of digoxigenin labeling of the cDNA probe were monitored by polyacrylamide gel electrophoresis, followed by transfer to nylon membrane and chemiluminescent detection. The digoxigenin-labeled cDNA products were further evaluated by their hybridization performance on HDFAs.
We evaluated three RT enzymes: Moloney murine leukemia virus (MMLV) RT, SuperScript II RT, and avian myeloblastosis virus (AMV) RT. Both MMLV RT and AMV RT have RNase H activity, whereas SuperScript II RT is a recombinant MMLV RT that lacks RNase H activity. Representative results are shown in Figure 1. The yield of digoxigenin-labeled cDNA synthesized by either MMLV RT or SuperScript II RT was consistently two- to fourfold higher than that synthesized by AMV RT (Figure 1, Lanes 24). Both MMLV RT and SuperScript II RT synthesized similar amounts of long cDNA, but SuperScript II RT produced more short products between 300 and 400 bases. The yield and size of digoxigenin-labeled cDNA synthesized by SuperScript II RT was similar whether the reaction occurred at 37C or 42C (Figure 1, Lanes 6 and 7).
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We tested oligo(dT) and random hexamer oligonucleotides either alone or in combination as primers for SuperScript II RT-mediated cDNA synthesis. As shown in Figure 1 (Lanes 46), effective priming and cDNA synthesis occurred with oligo(dT) primers, as well as with oligo(dT) in combination with random hexamers. Random hexamers alone were less effective primers.
We also examined the effect of varying the nucleotide concentration during cDNA synthesis, as well as the ratio of unlabeled to labeled dTTP. Equimolar deoxynucleotide concentrations of 0.5 mM, 0.2 mM, and 0.05 mM (close to Km of RT enzymes) were tested. Yield and size distribution were optimal with the 0.5 mM concentration (not shown). Changing the ratio of dTTP to digoxigenindUTP from 0.65:0.35 to 0.75:0.25 had no noticeable effect on cDNA synthesis (not shown).
Hybridization to High-density Filter Arrays
We evaluated the effectiveness of the cDNA labeling procedure by the results of hybridization to a commercially produced HDFA. The Atlas human cDNA expression array I (Clontech) has 588 cDNA targets spotted in duplicate as well as specificity control targets (negative control). To be useful, the results obtained must be at least equivalent to those illustrated by the manufacturer of the arrays using 32P-labeled probes and autoradiographic exposure in terms of the pattern and intensity of signals, background, and specificity.
For optimal detection of specifically hybridized digoxigenin probes, we followed the digoxigenin manufacturer's (Boehringer Mannheim) guidelines for hybridization solution and conjugate concentration and chose the most sensitive chemiluminescent substrate available, CDP-Star. In pilot experiments, we determined the optimal hybridization temperature and wash stringency conditions. Our prior experience with detection of digoxigenin probes indicated that additional blocking before detection with the antibodyalkaline phosphatase conjugate allowed minimal background, even with very long exposures.
Products prepared using the labeling conditions illustrated in Figure 1 were compared in a series of hybridizations to HDFAs (not shown). Because of poor yields as evaluated by polyacrylamide gel electrophoresis (Figure 1), AMV RT products were not tested by hybridization. The results of the cDNA digoxigenin labeling (size and incorporation of label) were most consistent with SuperScript II RT and, in general, the labeling reactions that included both high and low molecular weight products (illustrated as tail between arrows in Figure 1) gave the best combination of sensitivity (numbers of positive hybridization spots) and specificity (low background and absence of signal in negative control dots). On the basis of these pilot hybridizations, we selected SuperScript II RT labeling at 42C with oligo(dT) and random primers as the optimal method for preparing digoxigenin-labeled cDNA.
Using the optimized RT protocol to label placental poly (A)+ RNA, the results of chemiluminescent analysis of gene expression on the Atlas HDFA were identical to the autoradiographic results illustrated by the manufacturer. The influence of probe concentration was evaluated by comparing the hybridization results using either one third or the entire cDNA labeling reaction (Figure 2). Both probe concentrations could be used without increasing background or nonspecific signal to negative control spots (Figure 2, boxed area). At short exposure times, the higher probe concentration did detect expression of more genes than the lower probe concentration. However, at 1 hr of exposure, both probe concentrations produced similar expression profiles. In addition, the hybridization solution containing the probe could be reused to probe another HDFA without significant loss of signal or change in expression profile (not shown).
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To verify that the digoxigenin-labeled cDNA reflects the relative mRNA diversity in the original sample, we produced probes from commercially prepared placental poly(A)+ RNA and from poly(A)+ RNA isolated from monolayer cultures of normal foreskin keratinocytes (NFKc). The expression profiles obtained on HDFAs were distinct (Figure 3), demonstrating that placental and keratinocyte gene expression differences were reflected in the digoxigenin-labeled cDNA probe produced from each sample.
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Detection Limit for Low-abundance mRNA
Significant functional differences among samples may be attributable to differences in very low-abundance messages. Therefore, it is important to determine the detection limit of the cDNA labeling and hybridization procedure for rare transcripts. To test this, serial dilutions of human leukophysin mRNA was prepared in a background of D. melanogaster embryo mRNA. The cDNA probe produced from 1 µg of each RNA mixture was then hybridized to a nylon membrane spotted with human leukophysin cDNA and negative DNA targets (10 ng each, identical to the concentration of cDNA targets on the Atlas Array). The detection limit for leukophysin mRNA was at a dilution of 1:100,000 (w/w) in Drosophila mRNA (Figure 4). Therefore, chemiluminescent analysis of gene expression on HDFAs using this protocol will detect transcripts that constitute 0.001% of the total mRNA in a sample.
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Discussion |
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This is the first detailed report of optimized conditions for chemiluminescent analysis of gene expression using HDFA. We demonstrate that the method yields results comparable to or exceeding those obtained with autoradiography. In addition, results require shorter exposure times and the digoxigenin method permits storage and reuse of the hybridization solution. Importantly, we also demonstrate the ability of this approach to detect the presence of very low-abundance messages (115 transcripts/cell).
As anticipated, the conditions for cDNA synthesis influenced the results. We determined conditions that yielded the optimal results in terms of reproducibility, yield, size, and hybridization efficiency. In addition, we found that cDNA synthesis could be easily monitored by denaturing polyacrylamide gel electrophoresis, transfer to nylon, and chemiluminescent detection. Digoxigenin incorporation monitored using Dig-Quantification Strips (Boehringer Mannheim), did not give reliable estimates of synthesis efficiency. After digoxigenin labeling, the cDNA probe is used without further purification, eliminating one step that could contribute to nonreproducible loss of mRNA representation. Separation of unincorporated nucleotides and probe purification by ethanol precipitation or spin columns did not improve background and did not increase the signal.
SuperScript II RT was designed to eliminate the RNase H activity and, for radioactive synthesis, it is reported to be superior to MMLV RT in terms of yield and production of full-length cDNAs (
We demonstrated the success of chemiluminescent analysis of gene expression on HDFAs in several ways. Using the same commercial source of placental poly(A)+ RNA, our results using a 1-hr exposure exceed those achieved by the HDFA manufacturer using radiolabeled probes and a 37-day exposure. The HDFAs' expression profiles of cDNA prepared from two different sources (placenta and keratinocytes) reflected anticipated differences in the mRNA from these samples. The chemiluminescent detection of the 1:100,000 dilution (w/w) of leukophysin mRNA in the complex background of Drosophila mRNA is 10- to 25-fold more sensitive than that reported for radioactive detection (1:10,0001:20,000 w/w) on HDFAs (
Our approach to chemiluminescent analysis of gene expression on HDFAs focused on optimization of labeling, hybridization, and detection parameters using film. For affinity labels, detection is indirect and enzymatic generation of signal can produce nonlinear results. This nonlinearity, combined with the limited dynamic range of film, could compromise the usefulness of this approach to compare expression profiles between samples. These initial experiments on HDFAs indicate that chemiluminescent analysis of gene expression is within the dynamic range of film and will be amenable to quantification with chemiluminescent imaging devices. The chemiluminescent approach requires strict conditions for detection to achieve the low backgrounds and high sensitivity reported here. Compared with radiolabeling methods, the detection steps are longer and more complicated. In addition, the HDFA cannot be stripped and reprobed with another digoxigenin-labeled cDNA probe. For our work, this limitation is more than overcome by the ability to reuse the labeled cDNA probe to hybridize to a different HDFA, available from Clontech or other manufacturers, that includes additional cDNA targets. This approach to cDNA labeling could also be applied to other techniques, such as screening of cDNA libraries by colony hybridization and differential display PCR.
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Acknowledgments |
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Supported in part by appointments (MSR and IMD) to the Research Participation Program at the Centers for Disease Control and Prevention, National Center for Infectious Diseases, Division of Viral and Rickettsial Diseases, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the CDC.
We wish to thank Daisy Lee for preparation of the monolayer cultures of neonatal foreskin keratinocytes.
Use of trade names and commercial sources does not imply endorsement by the CDC or the US Department of Health and Human Services.
Received for publication August 14, 1998; accepted October 22, 1998.
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