REVIEW ARTICLE
Methods in molecular cardiology: microarray
technology
B. van den Bosch, PA. Doevendans, D. Lips, H.J.M. Smeets
It has become more and more evident that changes in expression levels of genes can play an important role in cardiovascular diseases. Specific gene expression profiles may explain, for example, the pathophysiology of myocardial hypertrophy and pump failure and may provide clues for therapeutic interventions. Knowledge of gene expression patterns can also be applied for diagnostic and prognostic purposes, in which differences in gene activity can be used for classification. DNA microarray technology has become the method of choice to simultaneously study the expression of many different genes in a single assay. Each microarray contains many thousands of different DNA sequences attached to a glass slide. The amount of messenger RNA, which is a measure of gene activity, is compared for each gene on the mkcroarray by labelling the mRNAwith different fluorescently labelled nudeotides (Cy3 or Cy5) for the test and reference samples. After hybridisation to the microarray the relative amounts of a particular gene transcript in the two samples can be determined by measuring the signal intensities for the fluorescent groups (Cy3 and Cy5) and calculating signal ratios. This paper describes the development ofin-house microarray technology, using commercially available cDNA collections. Several technical approaches will be compared and an overview of B. van don Bosch. Department of Genetics and Cell Biology, Cardiovascular Research Institute, P0 Box 5800, 6202 AZ Maastricht. P.A. Doevendans. Department of Cardiology, Heart and Lung Centre, PO Box 85500, 3508 GA Utrecht. ICIN, P0 Box 19258, 3501 DG Utrecht D. Ups. Department of Cardiology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht.
H.J.M. Snats. Department of Genetics and Cell Biology, Genome Centre, P0 Box 5800, 6202 AZ Maastricht. Address for correspondence: H.J.M. Smeets. E-mail: [email protected]
Nechrlands Hear Journal, Volume 11, Number 5, May 2003
the pitfalls and possibilities will be presented. The technology will be explained in the context of our project to determine gene expression differences between normal, hypertrophic and failing heart. (Net/ HeartJ2003;1 1:213-20.)
Keywords: microarray technology, molecular cardiology Oenes encoded in the DNA are transcribed into vmessenger RNA (mRNA), which is then translated into functional proteins (figure 1).' Microarrays detect gene expression (mRNA expression) levels by measuring the hybridisation of mRNA present in the tissue of interest to many thousands of genes immobilised on a glass surface (the array). Each single spot on the microarray represents a different gene. The double-stranded DNA has to be single stranded when deposited on the slides. In this way, the single-stranded DNA on the array is able to bind to complementary cDNA, to which the mRNA is converted. Both the test and reference mRNA samples are reverse transcribed into cDNA using oligo-(dT) priming and fluorescently labelled nucleotides are incorporated. The labelled cDNAs are then mixed together and hybridised to the microarray. After washing, the microarray is scanned to determine the amount offluorescence for each spotted gene. Since we know which gene each spot represents, and the cDNA (representing the mRNA) only hybridises to the gene that encodes it, we can determine the expression level of every individual gene. The fluorescence at each spot thus represents the expression level of the particular gene. Because one probe is labelled with Cy3 (emits green fluorescence at 532 nm) and the other is labelled with Cy5 (emits red fluorescence at 635 nm), quantitative comparison ofgene expression in both samples is possible. The ratio of Cy5 signal to Cy3 signal can be calculated for each spot on the array and indicates the ratio of gene expression (figure 2).'
Manufacturing of DNA microarays A number of techniques have been developed for manufacturing DNA microarrays. One of the ap213
Methods in molecular cardiology: microarray technology
. _~ ~.M_
Figure 2. Principle of microarray technology. cDNA probes are prepared by reverse transcrtiption ofmRNA samples to cDNA with fluorescently labelled nucleotides (Cy3/Cy5). Afterrhybridisation, slides are scanned and signal intensitiesfor the fluorescent groups are measured. The ratio ofCy5 signal to Cy3 signal is cakulated for each spot on the array, indicating the ratio ofgene expression between normal and bypertrophic tinue for that gene.1
Figure 1. Transcription of DNA into mRNA and translocation into proteins. Since we know which gene each spot represents, and the cDNA (reprsenting the mRNA) only hybridisesto thegene that encodes it we can determine the expzression level ofevery individual gene. Thefluorecence ateachspotthus represents the expression level oftheparticulargene. Because one probe is labelled with Cy3 (emits green fluorescence at 532 nm) and the other is lablled with Cy5 (emits redfluorescence at 635 nm), quantitative comparison of gene expression in both samples is possible.
(figure 3a)2 consists of an open ring with a pin centred above. When the ring is dipped into the solution and lifted, it withdraws an aliquot of DNA solution. To spot the sample, the pin is driven through the ring and 50 pl of product is deposited onto the slides. Another way of dispensing the DNA onto the slide is using 'split' pins (figure 3b)3 which are capable of a multidispensing mode of operation. The sample is transferred passively by surface tension as the pin tip touches the slide surface. There are different kinds of robotic systems for microarraying available.4 The Affymetrix Genetic Microsystems (GMS) 417 arrayer5 uses the pinand-ring system, while the MicroGrid II (Biorobotics) can use the split-pin system.6
proaches is called DNA micro-dispensing. Small quantities of DNA solution (with a volume of approximately 50 pl) are dispensed onto a solid surface. Products can be spotted onto the slides using pins, pins and rings, or split pins. The pin-and-ring system
DNA molecules can be spotted onto different kinds of glass surfaces (Telechem, poly-L-lysine [Sigma], aminosilane coated [Sigma, Corning]). Poly-L-lysinecoated slides are commonly used in combination with
A
The Pin-and-Rin a
g
_ . ... .. -------.....1
Fiyure 3. Two ways ofdoiting DNA solutions onto aghus slide. A. pin picks up the DNA solution and by driving the pin thugb the ring, the DNA is deposited onto the dides. B: vSplitpins transferthe sampk pasivelybysurfiae tenion as the pin tip tocbes the slide surfac. The
214
Netherlands Heart Journal, Volume 11, Number 5, May 2003
..
.'^,t=..,._F6E.e>..,-_._
Methods in molecular cardiology: microarray technology
1 1
.
2
3
MI 1 2 3 4 5 6 7 8 9 1011 121314 Fiqumr PCRframentsofcDNA ckmel ACknwftwm Ik 4k at 7w total t consists of40pls (eac containing 96 dones). Produc from pit ares/sown. Teprodu are around 500 bp ins/w. Lanes
4
5
2
..
qantification ofprduc andsive demination (Eurogntec). 71e markerprodces a patn nine regularly sd band4 rangin fprmlOOtol000p. 71* 100and00bpbandsbaabai/riei than tid otieto allow quik and easy identifiati. BC at cnis dxl5ksklxi ofl59plat (eawccontaininW 96cdmns). 7Tb sie of poduct vary from 0.5 to 3 kb. Columns I and 2 represnt two difrnt plate; of whbic 80 clones in tal are shn n (8 donsper lan). DiQfrncsinPCR efficicy an heosred (lae Ifromplatelforesmp). p4Alsosonsecontaminated fwntsens are present; containing more tan one produc as is indicatedbydxarrow (plate 2 lanem4 diwfnstfragsent).
.... t.
Methods in molecular cardiology: microarray
technology
B. van den Bosch, PA. Doevendans, D. Lips, H.J.M. Smeets
It has become more and more evident that changes in expression levels of genes can play an important role in cardiovascular diseases. Specific gene expression profiles may explain, for example, the pathophysiology of myocardial hypertrophy and pump failure and may provide clues for therapeutic interventions. Knowledge of gene expression patterns can also be applied for diagnostic and prognostic purposes, in which differences in gene activity can be used for classification. DNA microarray technology has become the method of choice to simultaneously study the expression of many different genes in a single assay. Each microarray contains many thousands of different DNA sequences attached to a glass slide. The amount of messenger RNA, which is a measure of gene activity, is compared for each gene on the mkcroarray by labelling the mRNAwith different fluorescently labelled nudeotides (Cy3 or Cy5) for the test and reference samples. After hybridisation to the microarray the relative amounts of a particular gene transcript in the two samples can be determined by measuring the signal intensities for the fluorescent groups (Cy3 and Cy5) and calculating signal ratios. This paper describes the development ofin-house microarray technology, using commercially available cDNA collections. Several technical approaches will be compared and an overview of B. van don Bosch. Department of Genetics and Cell Biology, Cardiovascular Research Institute, P0 Box 5800, 6202 AZ Maastricht. P.A. Doevendans. Department of Cardiology, Heart and Lung Centre, PO Box 85500, 3508 GA Utrecht. ICIN, P0 Box 19258, 3501 DG Utrecht D. Ups. Department of Cardiology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht.
H.J.M. Snats. Department of Genetics and Cell Biology, Genome Centre, P0 Box 5800, 6202 AZ Maastricht. Address for correspondence: H.J.M. Smeets. E-mail: [email protected]
Nechrlands Hear Journal, Volume 11, Number 5, May 2003
the pitfalls and possibilities will be presented. The technology will be explained in the context of our project to determine gene expression differences between normal, hypertrophic and failing heart. (Net/ HeartJ2003;1 1:213-20.)
Keywords: microarray technology, molecular cardiology Oenes encoded in the DNA are transcribed into vmessenger RNA (mRNA), which is then translated into functional proteins (figure 1).' Microarrays detect gene expression (mRNA expression) levels by measuring the hybridisation of mRNA present in the tissue of interest to many thousands of genes immobilised on a glass surface (the array). Each single spot on the microarray represents a different gene. The double-stranded DNA has to be single stranded when deposited on the slides. In this way, the single-stranded DNA on the array is able to bind to complementary cDNA, to which the mRNA is converted. Both the test and reference mRNA samples are reverse transcribed into cDNA using oligo-(dT) priming and fluorescently labelled nucleotides are incorporated. The labelled cDNAs are then mixed together and hybridised to the microarray. After washing, the microarray is scanned to determine the amount offluorescence for each spotted gene. Since we know which gene each spot represents, and the cDNA (representing the mRNA) only hybridises to the gene that encodes it, we can determine the expression level of every individual gene. The fluorescence at each spot thus represents the expression level of the particular gene. Because one probe is labelled with Cy3 (emits green fluorescence at 532 nm) and the other is labelled with Cy5 (emits red fluorescence at 635 nm), quantitative comparison ofgene expression in both samples is possible. The ratio of Cy5 signal to Cy3 signal can be calculated for each spot on the array and indicates the ratio of gene expression (figure 2).'
Manufacturing of DNA microarays A number of techniques have been developed for manufacturing DNA microarrays. One of the ap213
Methods in molecular cardiology: microarray technology
. _~ ~.M_
Figure 2. Principle of microarray technology. cDNA probes are prepared by reverse transcrtiption ofmRNA samples to cDNA with fluorescently labelled nucleotides (Cy3/Cy5). Afterrhybridisation, slides are scanned and signal intensitiesfor the fluorescent groups are measured. The ratio ofCy5 signal to Cy3 signal is cakulated for each spot on the array, indicating the ratio ofgene expression between normal and bypertrophic tinue for that gene.1
Figure 1. Transcription of DNA into mRNA and translocation into proteins. Since we know which gene each spot represents, and the cDNA (reprsenting the mRNA) only hybridisesto thegene that encodes it we can determine the expzression level ofevery individual gene. Thefluorecence ateachspotthus represents the expression level oftheparticulargene. Because one probe is labelled with Cy3 (emits green fluorescence at 532 nm) and the other is lablled with Cy5 (emits redfluorescence at 635 nm), quantitative comparison of gene expression in both samples is possible.
(figure 3a)2 consists of an open ring with a pin centred above. When the ring is dipped into the solution and lifted, it withdraws an aliquot of DNA solution. To spot the sample, the pin is driven through the ring and 50 pl of product is deposited onto the slides. Another way of dispensing the DNA onto the slide is using 'split' pins (figure 3b)3 which are capable of a multidispensing mode of operation. The sample is transferred passively by surface tension as the pin tip touches the slide surface. There are different kinds of robotic systems for microarraying available.4 The Affymetrix Genetic Microsystems (GMS) 417 arrayer5 uses the pinand-ring system, while the MicroGrid II (Biorobotics) can use the split-pin system.6
proaches is called DNA micro-dispensing. Small quantities of DNA solution (with a volume of approximately 50 pl) are dispensed onto a solid surface. Products can be spotted onto the slides using pins, pins and rings, or split pins. The pin-and-ring system
DNA molecules can be spotted onto different kinds of glass surfaces (Telechem, poly-L-lysine [Sigma], aminosilane coated [Sigma, Corning]). Poly-L-lysinecoated slides are commonly used in combination with
A
The Pin-and-Rin a
g
_ . ... .. -------.....1
Fiyure 3. Two ways ofdoiting DNA solutions onto aghus slide. A. pin picks up the DNA solution and by driving the pin thugb the ring, the DNA is deposited onto the dides. B: vSplitpins transferthe sampk pasivelybysurfiae tenion as the pin tip tocbes the slide surfac. The
214
Netherlands Heart Journal, Volume 11, Number 5, May 2003
..
.'^,t=..,._F6E.e>..,-_._
Methods in molecular cardiology: microarray technology
1 1
.
2
3
MI 1 2 3 4 5 6 7 8 9 1011 121314 Fiqumr PCRframentsofcDNA ckmel ACknwftwm Ik 4k at 7w total t consists of40pls (eac containing 96 dones). Produc from pit ares/sown. Teprodu are around 500 bp ins/w. Lanes
4
5
2
..
qantification ofprduc andsive demination (Eurogntec). 71e markerprodces a patn nine regularly sd band4 rangin fprmlOOtol000p. 71* 100and00bpbandsbaabai/riei than tid otieto allow quik and easy identifiati. BC at cnis dxl5ksklxi ofl59plat (eawccontaininW 96cdmns). 7Tb sie of poduct vary from 0.5 to 3 kb. Columns I and 2 represnt two difrnt plate; of whbic 80 clones in tal are shn n (8 donsper lan). DiQfrncsinPCR efficicy an heosred (lae Ifromplatelforesmp). p4Alsosonsecontaminated fwntsens are present; containing more tan one produc as is indicatedbydxarrow (plate 2 lanem4 diwfnstfragsent).
.... t.
