High throughput drug screening by direct RNA profiling on DNA sensors

Analytica Chimica Acta 469 (2002) 141–148

High throughput drug screening by direct RNAprofiling on DNA sensors

Rahul Mitraa,b,∗, Tom Powdrillb, Michael E. Hogana,b,c

a Genomics USA Inc., 3623 Avey Court, Pearland, TX 77584, USAb Advanced Technologies for Clinical Genomics, T428 Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

c Genometrix Incorporated, 3608 Research Forest Drive, Suite B7, The Woodlands, TX 77381, USA

Received 10 August 2001; received in revised form 18 April 2002; accepted 14 May 2002

Abstract

The feasibility of DNA microarray sensor technology as a routine technique of molecular pharmacology to perform highthroughput drug screening and the advantages of directly labeled RNA for a high throughput experiment are presented inthis paper. A novel, single-step direct chemical labeling method for DNA microarray target samples has been developed toreduce the sample amount, cost, time and error of the experiment by eliminating the need for enzyme mediated labeling.Reproducibility of the data for high throughput drug screening is demonstrated by monitoring differential gene expression ofa set of 45 gene targets involved in the genotoxic stress response pathways.© 2002 Elsevier Science B.V. All rights reserved.

Keywords: Chemical labeling; Microarray; High throughput; Drug screening

1. Introduction

DNA microarray technology provides a unique wayto monitor the simultaneous gene expression patterns,identify single nucleotide polymorphisms, and geneticidentification[1,2]. The sensitivity and robustness ofthis highly parallel DNA sensor technology dependson the fabrication methodologies, choice of the ma-terials, and finally sample labeling. Of the covalentlyattached arrays, two types of arrays—cDNA andoligonucleotide, are currently used. The cDNA arraysuse a double stranded clone as a probe for each gene.This reduces the signal due to redundancy in the se-quence, double stranded nature of the clone, decreasein the density of the probe on the surface, and varia-

∗ Corresponding author. Fax:+1-281-489-9724.E-mail address: [email protected] (R. Mitra).

tions in length of the cDNA probes. Oligonuclecotidearrays minimize the redundancy of information be-cause they are much shorter in length. Hence, a higherdensity of the probe can be achieved. Careful designcould increase the uniformity of binding energy anddecrease the complexity of hybridization.

The other concern with the array technology is thetype of the target and the labeling method. Traditionalmethods of labeling the target[3,4] involve enzymaticmanipulations, usually done at 37–42◦C, where mostof the nucleic acid polymers take up some sort ofsecondary structure, which leads to a bias in thelabeling of the target. Further, a loss of target cannotbe ruled out during the purification steps, followingan enzymatic labeling. In order to measure changesin gene expression with high sensitivity, it is not onlyessential to create a DNA microarray sensor with highprecision, but also the target-labeling methods need

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142 R. Mitra et al. / Analytica Chimica Acta 469 (2002) 141–148

also be extremely accurate. Novel methods in theDNA microarray field are improving reproducibility,reducing the time and costs associated with the manu-facture and subsequent analyses of the overwhelmingamount of the data. This has led to an increase in thehigh throughput screening of different pharmaceuti-cal agent-treated tissue, and tumor samples. Amountand structural integrity of the target become criticalfactors in the success of a high throughput screeningexperiments on DNA microarrays. Hence a methodof target labeling that reduces the manipulation andallows for signal amplification by secondary meanswould be useful to improve the high throughputscreening on DNA microarrays.

We demonstrate one of the approaches to tackle theabove problems by the use of a 45-element DNA mi-croarray to achieve reproducible data for a known setof genes involved in the cellular physiological stressresponse pathways. Using a direct RNA chemical la-beling methodology, we show that bias in the targetpreparation could be eliminated and improve the tar-get requirements significantly. We show this by usinga fluorescent hapten (digoxigenin) to label cDNA andtotal RNA, enzymatically and chemically, and screena drug 1-�-d-arabinofuranosylcytosine (AraC), on theof apoptotic pathway[5] in human myeloid leukemiacells (HL60).

2. Materials and methods

2.1. Microarray design

Forty-five unique 20mer synthetic sequences(probes) representing the genes related to the cellularphysiological stress response, cell cycle regulationand house keeping were arrayed on a glass slide inthe 16-well format as shown inFig. 1. This set ofgenes is well studied in the context of drug action andapoptosis using standard molecular biology methodslike fluorescence activated cell sorting (FACS) DNAapoptotic laddering and cell viability. Thus, data existfor comparison and validation of the method pre-sented in this paper. The probes were designed byselecting unique sequences from the regions proximalto the 3′-end of the cDNAs represented in the genomesequence databases. The length was adjusted to 20bases to provide for equal free energy of binding.

A thorough sequence alignment using Basic LocalAlignment Search Tool (BLAST)[6] was performedto ascertain the uniqueness of the probes. Oligonu-cleotides purchased from Midland Certified ReagentCompany (Midland, TX, USA) were provided withmass spectrometric profiles to assure their purity.

2.2. Microarray printing

DNA microarrays were printed at Genometrix In-corporated (The Woodlands, TX, USA), using pro-prietary robotic microcapillary deposition. Duplicatecopies of probes along with�-actin, glyceraldehyde3-phosphate dehydrogenase (GAPDH) and alignmentmarkers, in the anti-sense orientation for the cDNAtargets (antisense-microarray) and in the sense ori-entation (sense-microarray) for mRNA, were printedon epoxysilane surfaces. The location of the align-ment markers was changed in the sense-microarraysfor easier identification. DNA microarrays were storedat 4◦C until their use.

2.3. Cell culture and drug treatment

HL60 cells (ATCC, Manassas, VA, USA) weregrown in Iscove’s modified Dulbecco’s medium with4 mM l-glutamine adjusted to contain 1.5 g/l sodiumbicarbonate and 20% fetal bovine serum. A total of100 U/ml penicillin and 100�g/ml streptomycin wereadded to the medium, prior to the drug treatment. Cellviability was determined by trypan blue exclusionand exceeded 95% in all instances. Cultures wereseeded at 2× 105 viable cells/ml and subcultured at1 × 106 cells/ml as suggested by the vendor (ATCC).AraC (Sigma), purchased as powder, was diluted inthe medium without serum to 1× 10−6 M and addeddirectly to the cultures. Cell aliquots (5× 106) treatedwith AraC were collected at different time points forthe preparation of total RNA, mRNA, genomic DNAand FACS analysis.

2.4. RNA extraction

Total cellular RNA was extracted by a modi-fied guanidine-isothiocyanate technique (Qiagen,Valencia, CA, USA). The quality and the concentra-tion of the RNA were checked using both spectromet-ric and dye tests (Invitrogen, Carlsbad, CA, USA).

R. Mitra et al. / Analytica Chimica Acta 469 (2002) 141–148 143

Fig. 1. Design of the DNA microarray is shown with the map of its 45 elements and markers are shown in the inset. Each slide contained16 wells with one array in each well. The close-up of one of the wells in the circle shows the map of the genes, each printed in duplicate,in the array.


2.5. cDNA preparation and labeling

The mRNA was converted to cDNA incorporatingthe label, digoxigenin, enzymatically, using the corre-sponding labeled digoxigenin-11-2′-deoxy-uridine-5′-triphosphate, dig-dUTP (Roche, Basel, Switzerland)in a standard reverse transcriptase reaction. Briefly,5�g of total cellular RNA was reverse transcribedusing MMLV-RT (Clontech, Palo Alto, CA, USA).The concentration of the dTTP was adjusted inthe reaction to accommodate dig-dUTP at a finaldig-dUTP/dTTP ratio of 3:1. Following the reaction,the enzyme was heat inactivated and the cDNA pu-rified by sodium acetate/ethanol precipitation. Totalcellular RNA from human placenta (Clontech) waslabeled exactly as the HL60 samples and used as acontrol.

2.6. Chemical labeling of mRNA with digoxigenin

Total RNA was labeled with the digoxigenin-modified amino platinum derivative (dig-Pt, Roche)by heating a 1:1 mix of dig-Pt and total RNA, respec-tively, to 99◦C for 30 min, resulting in the hydrolysisof mRNA to approximately 100 bases, as ascer-tained by gel electrophoresis (Fig. 2). In addition,incorporation of the label was 1 in every 40 bases.The unreacted dig-Pt was quenched using EDTA. Itwas found that there was no need to further purify

Fig. 2. Agarose gel (2%) electrophoresis pattern of fragmentationof the RNA during labeling with dig-Pt at 99◦C. Lanes 1 and 6contain DNA size markers, lanes 2 and 4 show the unfragmentedRNA, and lanes 3 and 5 show fragmented RNA. The size of thefragmented RNA corresponds to 100 bases.

the dig-Pt labeled RNA. After testing concentrations(1�g to 50 ng), 100 ng of total RNA was used in eachof the hybridization to the DNA microarray. RNAfrom human placenta (a highly active tissue, andhence, represents an over expression of all mRNAs)was used as a control in these experiments to showthat the probes on the microarray bound to a differentsample and the sample used did not bias the results inany way.

2.7. Hybridization, imaging and analysis

DNA microarrays were washed in water and washedin a pre-hybridizing solution (150 mM sodium citrate(pH 7.0), 5× Denhardt’s solution (0.01% solutionof Ficoll, polyvinylpyrrolidone, and bovine serumalbumin in a 1:1:1 ratio), for 30 min in a humidi-fying chamber. Labeled mRNA and cDNA sampleswere suspended in the pre-hybridization solution.Digoxigenin labeled synthetic oligonucleotide targetscomplementary to the alignment markers were addedat a concentration of 1× 10−13 M to both cDNAand mRNA samples. Total hybridization volume wasmaintained at 30�l per well in a humidifying cham-ber, and carried out at room temperature for 8 h forcDNA and 3 h for mRNA.

Following the hybridization, the solution was re-moved by aspiration and then microarrays werewashed for 45 min with 125 mM sodium citrate (pH7.0) with three changes at 1, 15 and 30 min. Anti-digFab fragment (alkaline phosphatase conjugate) wasadded (1:1000) to recognize the hapten, digoxigenin.Development of UV-excitable fluorescence at theprobe addresses containing bound target was made byenzymatic cleavage of the ELF-anti-dig phosphatasesubstrate (Molecular Probes, Eugene, OR, USA). Themicroarrays were washed four times in a solution of0.002 M Tris, 0.01 M NaCl and 0.1% Tween-20, fol-lowed by two washes in MilliQ water and dried in anoven at 45◦C before imaging.

Imaging was carried out on Array Worx (AppliedPrecision, Issaquah, WA, USA) by exposing the DNAmicroarrays for 5 s and capturing the fluorescent im-ages using a CCD camera. The intensities from theimages were extracted using the programs providedby Array Worx. The spots from the images generatedwere then analyzed by imposing a grid correspond-ing to the DNA microarray map. The intensities of the


spots (printed in duplicate and arranged adjacent toeach other) were measured by calculating the centerof the mass intensity. The intensities were processedby subtracting the background, and normalizing to the�-actin and GAPDH. Twelve independent experimentsfor each time point for both cDNA and mRNA wereperformed and standard deviation was calculated foreach of the spot intensities.

2.8. DNA fragmentation—apoptotic ladder

Fragmentation of genomic DNA as a result ofapoptosis was observed by isolating genomic DNAat time points, 0, 2, 6, 8, 24 and 48 h. GenomicDNA was run on a 2% agarose gel and stained withethidium bromide to check the apoptotic ladderingeffect.

Fig. 3. (a) The differential gene expression patterns of the AraC-treated HL60 cells at time points 0, 4, 6 and 8 h, on an anti-sensemicroarray, using dig-dUTP labeled cDNA reverse transcribed from total cellular RNA as a target. The last column shows the profile ofthe over expressing genes from the developing tissue, human placenta. (b) The differential gene expression pattern of the AraC-treatedHL60 cells at the time points 0, 4, 6 and 8 h along with the human placenta control, on a sense microarray using dig-pt labeled mRNA.Each row represents a different experiment.

2.9. FACS analysis

The DNA content was measured using FACS foraliquots drawn at 0, 1, 2, 4, 6, 8, 12, 24, and 48 h, toestimate the population of the cells under-going apop-tosis. The cells were washed in 1× phosphate bufferedsaline (PBS) and fixed in 70% ethanol (aqueous) for12 h at 4◦C. Following the fixing, they were washedagain twice in 1× PBS, and propidium iodide wasadded to the cells.

3. Results

The design of the DNA microarray, and the map ofits 45 elements including the controls of housekeep-ing genes, such as�-actin andGAPDH are shown in


Fig. 1. In order to validate and demonstrate the use ofthe DNA microarrays to evaluate the cellular responseto the action of a pharmaceutical agent on its targets,we have chosen the well-documented HL60 cell lineand used AraC to induce apoptosis. By chemicallylabeling the mRNA with a platinum salt of the digox-igenin at an elevated temperature (99◦C) we haveeliminated the cDNA synthesis step, achieved both la-beling and fragmentation of the RNA. The hydrolysisof the RNA to an average size of 100 bases (Fig. 2)reduced the time taken for the binding to the probes

Fig. 4. Comparison of the labeling methods. (a) Array to array variation (CV= 0.032), shown by plotting the log intensities of twogroups of experiments (12 each) for time point at 0 h (target from untreated cells). (b). Comparison of enzymatically labeled cDNA (�) tochemically labeled RNA targets (�). Average log values of intensity of the microarray spots of the RNA targets (bound to the microarray)extracted from Ara-C treated samples for 4 h (y-axis) were plotted against average log values of intensity of microarray spots of the RNAtargets from untreated samples at 0 h (x-axis). In at-test,P-value was 0.042 at 99.9% confidence level and the correlation (R) was 0.97.

on the surface. Due to the high sensitivity of the la-bel, the amount of the sample for the total RNA wasreduced from 1000 to 100 ng per hybridization. Thecomparison of the results for the cDNA and mRNAare shown inFig. 3a and b, respectively.

The experiments were performed 12 times. The ob-served differences in the means for each of the probewere found to be insignificant with an average coeffi-cient of variance (CV) of 0.032 (Fig. 4a). Regressionanalysis of the data was done on a personal computerusing statistical and plotting program, Sigma plot. The


standard deviation for each data point was calculatedfrom n = 12 time points, and regression analysis wasperformed at 99.9% confidence levels. Human pla-centa RNA was used as a control in both the cDNAand mRNA experiments.

In 12 experiments of AraC induction of apoptosisfor each time point, differential gene expression pat-terns for 40 of the 45 markers (five markers were toolow in cDNA microarray experiments and were ig-nored) normalized to�-actin and GAPDH showed asimilar trend with sense microarrays for cDNA andanti-sense microarrays of mRNA (R = 0.97 andP =0.042 in t-test analysis;Fig. 4b). The general trendobserved was an induction of genotoxic stress relatedgenes at 2 h that persisted up to 4 h, a decline at 6 hand culminating by 8 h.

The markers analyzed here show a clear consistencywith their behavior reported in the literature. The ex-periments done were repeated 12 times to ascertaintheir reproducibility. Additional evidence for the apop-totic state of the cells was gathered by DNA fragmen-tation, which starts at 8 h and is complete by 48 h.

Over expression of bcl-2 has been shown to sup-press the initiation of apoptosis in response to a vari-ety of stimuli including anti-cancerous agents[7]. Thepredominance of the bax over bcl-2 was shown to en-hance apoptosis[8] in acute childhood leukemia. Ingeneral, if the bcl-2:bax� 1, the cells are guardedagainst apoptosis and if bcl2:bax 1 they are apop-totic. Our results for the bcl-2 and bax using bothcDNA and mRNA show that the level of the bcl-2falls upon the treatment with AraC, consistent with theobservations made earlier[8]. The induction of baxpeaks at 4 h and falls by 8 h.

Immediate early genesc-jun, jun-D andjun-B wereshown to be induced in response to AraC[9]. Ourresults are consistent with this observation.

The cell cycle related genes,cdk4, cyclin B, cyclinD1 persist once induced. The increased levels of cyclindependent kinase 4 and inappropriate re-entry into thecell cycle was shown to be a part of AraC inducedapoptosis.

Measurement of the DNA content and the cell cycleanalysis (data not shown) show that the sub-G popula-tion begins to increase from 1 h and peaks at 48 h (per-centage sub-G population at hours 1, 17.5; 2, 19.4; 4,24.6; 6, 32.4; 8, 35.7; 12, 35.3; 24, 25.4; 48, 41.9), cor-relating with the DNA fragmentation. Based on these

data, we conclude that the time points chosen to studythe differential gene expression patterns were appro-priate. All the observations consistent with the litera-ture point out that the DNA microarrays are well suitedfor the study of the action of the drug action on cells.

4. Discussion

4.1. High throughput screening—cDNA or mRNA?

Till date, enzymatic synthesis of cDNA has been themethod of choice for labeling RNA, due to the difficul-ties in labeling RNA populations directly. This indi-rect approach, although widely used, may not work ina high throughput experiment since it requires a largeamount of sample (∼1–50�g), increases cost, timeand probably loss of some sample during purificationsteps. One of the major issues in the use of cDNA asa target is the bias created in the reverse transcriptiondue to the secondary structure effects at 42◦C. Com-parison of the data from cDNAFig. 3aand total RNAFig. 3bshow a low or no signal for 5 out of 45 geneswhen cDNA is used instead of RNA. This we think isdue to the secondary structure effects on the target attemperatures of the reverse transcription reaction. Thelabeling method presented in this paper (performedat 99◦C) reduces the secondary structure and hence,ensures labeling of all of the targets.

4.2. Improvements in amount of sample, cost,error, time and design

By labeling the total RNA with dig-Pt at 99◦C,we achieved both hydrolysis to create target RNApopulations of uniform length and labeling at thesame time. This additionally reduces the free energycontribution due to the secondary structure for thebinding of the target and probe on the microarray.Since the unused label was quenched, there was noneed to purify the labeled product, thereby minimiz-ing error or loss of sample. Additionally, due to thehydrolysis of the RNA to shorter lengths the kineticsof the hybridization were altered reducing the timetaken for the mRNA hybridization to 5 h as opposedto 12–18 h required for a cDNA experiment. A shorterRNA target could overcome the problems involvedin binding—by reduction of secondary structure,


increase in discrimination between match and a mis-match, and probably due to the marginal increase instability of RNA–DNA hybrid.

Currently, most of the probe sequences chosen forthe DNA microarrays could be mapped to the 3′ re-gions of the genes. This is done in order to ensure therepresentation of the probe binding sequence in thereverse transcribed cDNA. Given the other constraintslike GC content, uniqueness, and secondary structure,it could be difficult to always find the probe sequencein the 3′ region of the gene. By the use of direct-labeledRNA populations as targets, the probe sequence is notrestricted to the 3′ region. It can be from any part of thegene, since every part of the mRNA is equally repre-sented in the directly labeled populations. This offersgreater freedom to design unique probes. It could alsoserve as an elegant internal control for the measure ofthe relative levels of gene expression by comparingbinding of targets to the probes from different regionsof the same gene. We have observed that by printingsequences from 3′- and 5′-ends of the gene, we wereable to clearly tell if any sequence was cross hybridiz-ing. In our study, using a p53(−) cell line, HL60, wefound some cross hybridization to the 3′-end probefor p53 (data not shown). Cross hybridization cannotbe ruled out in microarray experiments, but by usinga sequence from 5′-end we were able to check if thedirectly labeled mRNA, representing complementarytarget sequences from both 5′- and 3′-ends, was crosshybridizing to p53 probe. It was found that the p53probe sequence from the 5′-end was more robust andthe directly labeled mRNA did not bind to it, high-lighting another one of the advantages of using a di-rectly labeled RNA in a microarray experiment.

Our results show that in cases where the tissuesamples are difficult to obtain, the stringency, andreproducibility of the data is essential. The use ofchemically labeled mRNA reduces the cost, timerequired for the experiment and avoids the possibilityof error. Sample amount is reduced to 50 ng per wellof total RNA, which in this case translated to 500 pg ofmRNA.

5. Conclusions

We have demonstrated a novel method of labelingthe target RNA populations for DNA microarray ap-plications. The advantages offered by this one-steptechnique are reduction in the amount of samplefor each experiment, reduction of the loss of thesample, decrease in the bias and uniformity of thelabeling. The method presented here is better thanthe current enzymatic labeling methods and improvesthe speed and costs in a typical high throughputexperiment.

Acknowledgements

We thank Cynthia Edwards, Baylor College ofMedicine, for her help with the cell culturing andMini Kapoor, MD Anderson Cancer Center, for herassistance with the FACS analysis.

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High throughput drug screening by direct RNA profiling on DNA sensors