Comparison of two probe preparation methods using ...

RESEARCH REPORT

Comparison of two probe preparation methods using long oligonucleotide microarrays Fahd Al-Mulla, Raba’ Al-Tamimi, and Milad S. Bitar BioTechniques 37:827-833 (November 2004)

The use of oligonucleotides as a capture platform for microarray-based experiments is gaining popularity. Oligonucleotide-based microarrays involving various probe preparations have been compared by a number of researchers. Limited data are available, however, regarding the concordances and efficacies of various probe preparations on long oligonucleotide-based microarrays. Accordingly, the current investigation assesses two labeling methods, namely Atlas™ PowerScript™ fluorescent cDNA (random priming) and T7 in vitro transcription cRNA [poly(T) priming] labeling kits. Our data revealed that a high degree of reproducibility among the examined genes for each assay used with correlation coefficients of 0.93 and 0.94 for random priming and poly(T) priming, respectively. It is worthy of note, however, that when the two assaying methods were compared, the data showed a poor correlation coefficient. A confirmatory step involving real-time reverse transcription PCR (RT-PCR) of 18 selected genes favors the superiority of the cDNA fluorescent labeling over the T7 labeling method. Overall, the microarray results generated by the poly(T) priming methodology should be viewed cautiously even when high reproducibility is evident.

INTRODUCTION Microarray technology was first exploited in 1995 by Schena et al. (1). It is now a leading technology in large-scale gene expression profiling with an exponential increase in its use in biomedical research. The advantage of this technology is its ability to screen thousands of genes per experiment, as well as the parallel comparison of differentially labeled samples. This technology has proven itself to be highly valuable in deciphering the complexity and influences of genes and environmental factors on many diseases (2–5). The technology itself appears straightforward. However, its application is influenced by many factors. For example, the availability of several types of capture platforms and the availability of several labeling methodologies. Regarding the choice of a suitable capture platform, spotted oligonucleotide microarrays display several advantages over cDNA microarrays. These include the fact that only knowledge of gene sequences and no biological materials are required for

their synthesis. In addition, the oligonucleotides can be synthesized in ways to exclude homologous sequences between genes thus enhancing specificity. Also, different regions of a given gene can be targeted with different oligonucleotides allowing detailed mapping of splice variants and poly(A) transcripts. Long oligonucleotides, as with cDNAbased microarrays, lack the ability to discriminate between single nucleotide polymorphisms and mismatches. Therefore, longer oligonucleotides acquire more sensitivity with compromised specificity (6,7), a characteristic they share with cDNA-based microarrays. Indeed, recent data shows oligonucleotide-based microarrays to be equal in sensitivity to PCR or cDNAbased microarrays (8,9). Generally, results obtained from short oligonucleotides and cDNA-based microarrays correlate well (10–12). Nevertheless, recent reports have shown little correlation between different platforms, which could potentially limit the utilization of data from cross-platforms (13–15). It is worthy of note that it is not clear

whether the lack of correlations, in at least some of these studies, are attributed to differences in RNA, labeling methodologies, or the use of different platforms. This is compounded by the fact that only a few authors actually confirmed their expression results using alternative and independent techniques (13). Regarding RNA labeling, several methods are currently available. These range from synthesis of cDNA with direct labeling when RNA is abundant (16), to several-fold amplifications of RNA when its quantity is limited (17,18). The general consensus reported, albeit from limited investigators, is that microarray data generated from amplified RNA is comparable to nonamplified RNA. For example, Wang et al. (19) have applied 1–3 rounds of amplification and have found that the fidelity of amplified RNA from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA-based or poly(T) RNAbased arrays. Similarly, Scheidl et al.

Kuwait University, Safat, Kuwait Vol. 37, No. 5 (2004)

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RESEARCH REPORT (20) and others (10,21–23) have shown highly concordant results generated between amplified RNA and total RNA. In contrast, recent work has shown that gene expression profiling using amplified versus nonamplified RNA is not completely preserved and in some instances show poor correlations (15,24,25). Therefore, it is evident that there is a need for further detailed comparisons of data sets obtained from different labeling methodologies on identical platforms. With the availability of a large number of commercial kits, it is advisable to test and compare the results generated from several of these before choosing the appropriate one for a specific application. To that premise, two different probe preparation methods in the form of commercially available kits were tested, namely, BD Biosciences Clontech’s reverse transcription (cDNA; random priming) and Roche Applied Science’s T7 in vitro transcription [cRNA; poly(T) priming] (10). In this study, 80-nucleotide (nt) oligonucleotide-based microarrays were used as an intermediate microarray design that sustains appropriate levels of sensitivity and specificity. We aim to determine which labeling method yields more accurate and sensitive results on these platforms. MATERIALS AND METHODS Cell Culture and Total RNA Extraction Colo320 cells were cultured in supplemented RPMI-1640 medium [1% L-glutamine, 10% fetal bovine serum (FBS), and 1% antibacterialantimycotic agent supplements]. Cells were incubated at 37°C, in 95% humidity, and 5% CO2 saturation. Cells were harvested at 70% confluence and prepared for RNA extraction using TRIzol® (Life Technologies, Hamburg, Germany) according to the manufacturer’s criteria. Extracted RNA was quantified using spectrophotometry and analyzed for quality by 1% denaturing agarose gel electrophoresis. Adequate total RNA aliquots were DNase-treated for microarray probe preparation using a DNA-free kit (Ambion, Austin, TX, USA). 828 BioTechniques

cDNA Probe Preparation Method Total RNA was used to prepare microarray probes according to Atlas PowerScript Fluorescent Labeling kit manufacturer’s protocol (BD Biosciences Clontech, Palo Alto, CA, USA). In summary, a master mixture containing 2× first-strand buffer, 2× dNTP mixture (dCTP, dGTP, dATP, dTTP, aminoallyl-dUTP), 0.02 M dithiothreitol (DTT), 10 U/μL PowerScript reverse transcriptase, and deionized water was prepared. Five-microgram (7 μL) aliquots of normal colon total RNA (BD Biosciences Clontech) and Colo320 total RNA were prepared. Two microliters of random primer mixture and 1 μL cDNA synthesis control were added to RNA samples. RNA samples were incubated for 5 min at 70°C in a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA). Samples were then cooled at 37°C, and 10 μL master mixture were added. Samples were incubated at 37°C for 60 min and were later heated up to 70°C for 5 min. After cooling down at 37°C, 10 U/μL RNase H were added, and the reaction mixture was mixed well, vortex mixed, and incubated at 37°C for 15 min to degrade the RNA templates. To inhibit the reaction, 0.0125 M EDTA and 2 μL QuickClean™ (BD Biosciences Clontech) resin were added. The mixture was vortex mixed for 1 min and then centrifuged to collect the contents. The supernatant was transferred into a 0.22-μm spin filter tube and centrifuged at 12000× g for 1 min. Then, 2.2 μL 3 M sodium acetate and 55 μL cold absolute alcohol were added to the elutant and vortex mixed. The reaction mixture was left to precipitate for 2 h at -80°C. Centrifugation at 12000× g for 20 min to pellet cDNA was performed later. The pellet was washed with 70% alcohol, airdried, dissolved in 10 μL 2× fluorescent labeling buffer, and 0.5 μL of the coupling reaction oligonucleotide was added. Cy™3- and Cy5-free dyes (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) were dissolved in 45 μL dimethylsulfoxide (DMSO) to prepare a 5-mM stock. Ten microliters of each Cy dye were added to normal colon cDNA and Colo320 cDNA, respectively. Samples were incubated

at room temperature in the dark for 1 h to allow fluorescent dye coupling to amino acid-dUTPs. Samples were then precipitated at -80°C for 2 h with sodium acetate and absolute alcohol. The resultant pellet was washed with 70% alcohol and air-dried. The pellet was dissolved in 100 μL nuclease-free water (Ambion). Probe purification was performed using a QIAquick® PCR purification kit (Qiagen, Valencia, CA, USA). The amount of cDNA added to the microarray is determined according to the fluorescent dyes absorbance reading (Cy3 = 550 nm, Cy5 = 650 nm) using the following formula: Vopt (μL) = 1000 × OUλ/Aλ (where V = volume of probe to be added in μL, λ = fluorescent dye specific wavelength, and OUλ = optical units of probe at dye specific wavelength). Optimal Cy3 OU550 and Cy5 OU650 = 0.01. cRNA Probe Preparation Method The microarray cDNA synthesis kit (Roche Applied Science, Penzberg, Germany) was employed to synthesize double-stranded cDNA with incorporated T7 promoter sequence according to the manufacturer’s protocol. Ten micrograms Colo320 and normal RNA were mixed with 200 pmol T7 oligo(dT)24 primer, heated at 70°C for 10 min, and placed on ice immediately. Reverse transcriptase buffer (1×), 10 mM DTT, 50 U reverse transcriptase enzyme, and 1 mM each dNTP mixture were added to the RNA template mixture. The reaction mixture was incubated for 60 min at 42°C and put on ice to stop the reaction. A second master mixture containing 1× second-strand synthesis buffer, 0.1 mM of each dNTP, and 6.5 μL second-strand synthesis enzyme mixture (DNA polymerase I, Escherichia coli ligase, RNase H) was mixed gently with the first reaction mixture and incubated for 2 h at 16°C. Twenty units of T4 DNA polymerase were added to the mixture and incubated for 5 min at 16°C. The reaction was deactivated with 0.2 M EDTA. Template RNA was digested with 15 U RNase I. The reaction product was purified using a microarray target purification kit (Roche Applied Science). One microgram of Vol. 37, No. 5 (2004)

purified double-stranded DNA was dried in a vacuum and used as a template for the microarray T7 RNA target synthesis kit (Roche Applied Science). A dNTP mixture of 5 mM of each dNTP except dUTP was 3.75 mM and was added to the template along with 10 mM DTT, 1× transcription buffer, 3 μL transcription enzyme blend, and 1.25 mM Cy3- or Cy5-UTP. The mixture was incubated for 3 h at 37°C and stored on ice. Resultant cRNA probe was purified with the microarray target purification kit. Absorbance readings at 260, 550 (Cy3), and 650 (Cy5) were measured for samples, and according to the nucleic acids concentrations in the samples, a standard of 2.5 μg of each sample was mixed for hybridization. Probe Hybridization and Analysis Human 80-nt oligonucleotide microarray glass slides (BD Biosciences Clontech) were used. Four trial kit microarrays were used for reproducibility assessment and two 3.8-K oligoucleotide microarray slides. GlassHyb™ hybridization solution (2.1 mL; BD Biosciences Clontech) was prewarmed at 50°C in a 5-mL tube. Appropriate amounts of the two differentially labeled cDNA probes were mixed and added to the hybridization solution. Uniformly mixed hybridization solution was pipeted into the microarray slide chamber and left for an 18-h incubation at 50°C in the dark. The mixtures were gently rotated by hand three times during the 18-h hybridization. The same method was employed for cRNA probes, however, the cRNA probes were preheated at 95°C for 5 min and incubated on ice for 3 min prior to their hybridization. Microarray slide washing involves three washes. The first wash was with 22 mL of the GlassHyb wash solution. The second wash, a mixture of 2 mL GlassHyb wash solution and 20 mL 1× standard saline citrate (SSC), was prepared and repeated twice. The third wash was with 22 mL 0.1× SSC. The duration of each wash was 10 min at room temperature in the dark. The slides were air-dried in a vacuum in a vertical position. Oligonucleotide microarray glass slides were scanned using a Model LS200 laser scanner (Tecan, SalzVol. 37, No. 5 (2004)

Table 1. Overview of the Number of Genes Detected by Each Labeling Method Number of Genesa

Description Genes on the 3.8-K microarray Genes filtered out from all arrays (intensity below 0.01 threshold) Genes detected by the cDNA method Genes detected by the T7 method Genes detected by both methods with similar ratios Genes detected by both methods but with inconsistent ratios aPercent

3800 (100%) 3366 (88.5%) 285 (7.5%) 220 (5.8%) 100 (2.6%) 81 (2.1%)

of total is in parentheses.

berg, Austria). Data were analyzed using Array-Pro™ analyzer version 4.5 (Media Cybernetics, Silver Spring, MD, USA). Signal normalization was based on the median value of selected signal control cells of housekeeping genes. Signal intensities below 0.01 after normalization were omitted. Cy3/Cy5 signal ratios were calculated by the software. Threshold values for amplification and deletion ratios were >1.5 and 0.01 after normalization. The cRNA poly(T) priming method generated 401 genes. Only 181 genes were detected by the two methods. Of these, 100 genes (55%) had similar expression ratios, while the remaining 81 genes (45%) had different ratios between the two methods. The cRNA hybridized microarray had 220 (55%) genes that were absent from the cDNA hybridized microarray, while the cDNA hybridized microarray had 285 (61%) genes that were undetected by the cRNA hybridized array (Table 1). That the number of positive genes was small, inspection of each dot was confirmed visually. Expression data obtained from the two microarrays are compared in Figure 1. A poor correlation of 0.3 was obtained between the Colo320 Cy5-labeled cDNA from one array compared to the Colo320 Cy5-labeled cRNA from the other array (Figure 1A). This correlation was even lower (0.0045) when the data represented BioTechniques 829

RESEARCH REPORT by Cy5/Cy3 ratios for each method were plotted (Figure 1B). Because the same RNA batch and identical arrays were used, this unexpected result could have been generated by erroneous labeling. To test the reproducibility of labeling, each probe preparation method was repeated twice using another RNA batch and hybridized to four smaller microarray slides composed of 92 oligonucleotides. The

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92 dotted oligonucleotides correspond to genes expressed in various tissues but include genes that are universally expressed. Every two slides were normalized identically. Calculated correlation coefficient for cDNA probed microarrays was 0.93, and the correlation coefficient for cRNA probed microarrays was 0.92, indicating faithful reproducibility in both labeling methodologies (Figure 2, A and B). Nevertheless,

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�� Figure 1. Correlation scatterplots of two microarray platform signal intensities generated using the two-probe preparation methods. (A) Cy5 Colo320 cDNA versus Cy5 Colo320 cRNA resulted in poor correlation. (B) Cy5 Colo320/Cy3 normal colon cDNA ratio versus Cys5 Colo320/Cy3 normal colon cRNA ratio. 830 BioTechniques

of the 92 genes, 70 genes generated signals within the detectable threshold in the cDNA probe experiment, while only 21 genes were detected in the cRNA probe hybridizations. The sets of detected genes in the repeated experiments were consistent and had consistent ratios. It was obvious at this stage that the poly(T) priming was less sensitive than the random priming method. Attempts to increase laser gain did not result in higher detection or in improving the poor correlations between the methods. Similarly, fragmenting the cRNA, although not recommended by the manufacturer, but used by various other researchers (see References 6, 29, and 32), resulted in poorer correlation coefficient (data not shown). To assess the probe sizes generated by the two labeling kits, Cy3 cRNA and cDNA probes were run on 1% agarose denaturing gel. Interestingly, the two labeling methods produced probes mostly within the expected range of 396–2000 bases, although the cDNA labeling method generated much longer fragments (Figure 3). Because Cy5 and Cy3 dyes have previously been shown to have different incorporation kinetics and quenching susceptibility, which could bias the results, we labeled independent placental RNA with Cy5 and Cy3 dyes using random and poly(T) priming and have found that the different dyes had no or minor effects on the resultant ratios (data not shown). In an attempt to verify which labeling method reflects the true RNA population, RT-PCR was employed as an independent method on the original batch of extracted RNA. Eighteen genes were selected for this comparison. Four genes, namely, c-Myc, v-Ski, growth-associated protein 43, and sema domain all showed high expression ratios by both methodologies. Seven genes, transcription-like growth factor-1 (TFL-1), interferon γ receptor (INFγR), cyclin A2, patched homolog, serine/argenine repetitive matrix 2, nuclear factor like-1 (NFL-1), and fibroblast growth factor 6 (FGF-6) were overexpressed as detected by cDNA method alone. Four genes, jumping translocation break point, RNA-binding protein, ABO transferase A, and link guanine nucleotide exchange factor II Vol. 37, No. 5 (2004)

We set out to evaluate two microarray labeling kits that represent the two most widely used RNA labeling methods for microarray-based experiments. For hybridization, we chose long oligonucleotides platforms because they have been shown to be equal in their sensitivity to PCR probe and cDNA microarrays, with the added advantage of ease of manufacturing, a characteristic that is making them popular among researchers. The low correlation coefficient obtained from the large 3.8-K microarray was unexpected. A determination of microarray results reproducibility was performed. The cDNA synthesis probe preparation and the T7 in vitro transcription methods are two reproducible methods individually. The R2 values for resultant ratios in the duplicate experiments were 0.92 for cRNA and 0.93 for cDNA, respectively. This phenomenon of results reproducibility through repetitions of experiments under comparable conditions was used previously by many researchers on cDNA and short oligonucleotide microarrays and was assumed to be a measure of reliability of the labeling methodology (21–24). Our results caution against this qualitative assessment of reproducibility and should encourage further evaluation of generated data by independent methodologies, such as RT-PCR, Northern blot analysis, or Western blot analysis until a suitable, more robust wide-scanning method or a unified protocol is attainable. Careful review of the literature demonstrates that many microarray experiments using the poly(T) priming methodology have been published without further Vol. 37, No. 5 (2004)

using the RT-PCR technique. The RTPCR results would appear to favor the cDNA random priming method. All cDNA detected genes had similar expression profiles when screened by RT-PCR. The cRNA poly(T) priming method reported low sensitivity and specificity when compared to cDNA random priming method. However, there were at least two genes that were found overexpressed by the poly(T) priming method but not by the random

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cDNA Trial 2 Cy3/Cy5

DISCUSSION

validation of the results (21,26–29), while others have used qualitative validation in the form of reproducibility (6,10,18,20,24,30,31). Only a few have validated their results with other independent methods (11,20,22), and among these, some used the amplified RNA or cRNA material as the source for the validation process (20,32) or chose a few genes for confirmation of the expression results (22,33). We have confirmed our results with 18 genes

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showed overexpression by the cRNA method alone, and two genes, jagged 1 and cell cycle related kinase (CCRK), were assigned as underexpressed by the cDNA method, although the latter gene was assigned as overexpressed by the cRNA labeling method. RT-PCR normalized expression ratio results for the selected genes are illustrated in Figure 4. The resultant accuracy, sensitivity, and specificity were calculated for both labeling methods and are shown in Table 2.

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Figure 2. Reproducibility curves produced from duplicate microarray experiments for each probe preparation method. (A) The cDNA probes showed efficient reproducibility (R2 = 0.93), as did the cRNA probes (B) with an R2 of 0.92. BioTechniques 831

RESEARCH REPORT Table 2. Statistical Measurements for the Two Probe Preparation Methods Were Calculated Based on the Confirmatory RT-PCR Results Statistical Analysis

cDNA Probes (%)

cRNA Probes (%)

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Figure 3. Probes produced by the two labeling methods. One percent denaturing agarose gel of Cy3-labeled cRNA in lane 2 and Cy3labeled cDNA in lane 3. Intact total RNA is shown in lane 4. Lanes 1 and 5 are molecular weight markers.

priming method, and these were also confirmed by RT-PCR, indicating the usefulness of this method for detecting some transcripts (perhaps low transcript genes) (22,23). The overall low sensitivity of the poly(T) priming, however, curtails such an argument. It would have been advantageous to our study to know the region of the genes dotted on the array to equate it with the regions amplified by RT-PCR. However, we have found that obtaining such data from the commercial sources was impossible to achieve. Nevertheless, our findings of high accuracy, sensitivity, and specificity of the cDNA random priming method are inline with previously published work (16,34) and show that the oligonucleotides dotted could be equated to regions amplified by RT-PCR.

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We have not investigated the origin of the bias between the two labeling methods. There could be many reasons for such differences in expression profiling. Although we have controlled for laser gains and cRNA fragmentation, other cause such as the loss of substantial amounts of labeled cRNA probes through the intrinsic nucleolytic nature of T7 RNA polymerase is possible (32). However, in our case, this may have had minimal effect since the reactions were not prolonged for more than 3 h. Another plausible cause is that the cRNA probe generation method produces template-independent products in addition to amplifying target mRNA, thus potentially reducing the specific activity of the final products (18). In conclusion, the microarray results generated by the poly(T) priming methodology should be viewed cautiously even when high reproducibility is evident. Thus, many variables interfere with the microarray results, and every microarray technology requires tailored protocols determined from experience It is preferable, therefore, that quantitative rather than qualitative confirmational steps be taken to assess data generated from microarrays. ACKNOWLEDGMENTS

We thank Professor Samuel Olusi for his helpful comments on the manuscript. This work was funded in part by grant no. 99-07-07 from the Kuwait Foundation for the Advancement of Sciences and the Graduate College of Kuwait University.

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COMPETING INTEREST STATEMENT

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Figure 4. Histograms showing the expression ratios of 18 selected genes obtained from the two labeling methods and their corresponding real-time reverse transcription PCR (RT-PCR) results. Notice the erroneous expression ratios generated by the cRNA labeling method for cell cycle related kinase (CCRK), jagged 1, and link guanine nucleotide exchange factor II genes and its low sensitivity of detecting the stretch of genes from nuclear factor like-1 (NFL-1) to patched homolog. ABO transferase was undetectable by cDNA labeling and RT-PCR. 832 BioTechniques

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Received 21 January 2004; accepted 18 June 2004. Address correspondence to: Fahd Al-Mulla Kuwait University Faculty of Medicine Department of Pathology Molecular Pathology Laboratory P.O.Box 24923 Safat, Kuwait, 13110 e-mail: [email protected]

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