Microarray technique

Microarray -types, DNA chip, Principle and application of microarray, Preparation of DNA Chip, Affymetrix chip, microarray in genomics and proteomics, advantages and limitations of microarray

1. MICROARRAY TECHNIQUE ARUN CHACKO PhD SCHOLAR PLANT BREEDING & GENETICS

2. An array Orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. A DNA microarray ( DNA chip or biochip ) is a collection of microscopic DNA spots attached to a solid surface. An array experiment makes use of common assay systems such as microplates or standard blotting membranes.

3. History of Microarray 1965 : Gillespie and Spiegelman described methods for DNA blotting hybridization, in which DNA immobilized on a membrane can bind to complementary RNA or DNA strand through specific hybridization. 1975 : Southern blotting developed by E. M. Southern

4. Microarray technology evolved from Southern blotting. The concept of microarrays was first proposed in the late 1980s by Augenlicht and his colleagues. They spotted 4000 cDNA sequences on nitrocellulose membrane and used radioactive labeling to analyze differences in gene expression patterns among different types of colon tumors in various stages of malignancy.

5. 1982 : RNA was isolated from normal and cancer tissue of mice, cDNA synthesized, cloned on E. Coli and 378 colonies were arrayed. 1991 : Scientists at the California-based biotech company Affymetrix produce the first DNA chips.

6. 1995 : Quantitative monitoring of gene expression patterns with a complementary DNA microarray. 1995 :microarrays for gene expression profiling was done using complete eukaryotic genome ( Saccharomyces cerevisiae ) on a microarray chip.

7. The main applications of microarrays include: Comparative genome analysis (Healthy and infected cell) Functional genome analysis (to describe interactions between genes at different time point) Developing knowledge of gene function Discovery of drugs Diagnostics and genetic engineering Toxicological research ( Toxicogenomics )

8. Principle of Microarray technique mRNA : carries the genetic information from the cell nucleus to the cytoplasm for protein synthesis. These mRNAs synthesize the corresponding protein by translation. Indirectly by assessing the various mRNAs, we can assess the genetic information or the gene expression thus helps in the understanding of various processes behind every altered genetic expression.

9. The principle behind microarrays is hybridization between two DNA strands

10. Major steps in Microarray 1. Preparation of microarray 2. Preparation of labelled probes 3. Hybridization 4. Scanning, imaging and data analysis

11. PROBE TYPE ADVANTAGES DISADVANTAGES PCR Products Inexpensive Handling problems Hard to design to avoid cross- hybridization Unequal amplification Oligos Can be designed for many criteria Easy to handle Normalized concentrations Expensive A f fymetrix GeneChip High quality data Standardized arrays Fast to set up Multiple probes per gene Expensive Arrays available for limited number of species Methods for constructing the arrays:

12. Conventional methods can be used to produce the sequences (oligonucleotides), and these can then be printed directly onto the microscope slide (which is first overlaid with a coating that is positively charged). Microspotting Techniques

13. First used by AGILENT This is a non-contact process. Minute volumes of reagents are delivered to defined locations on the slide similar to ‘ ink-jet’ printing methods. A biochemical sample is loaded into a miniature nozzle equipped with a piezoelectric fitting (rectangles) and an electrical current is used to expel a precise amount of liquid from the jet onto the substrate . Piezoelectric Printing

14. After the first jetting step, the jet is washed and a second sample is loaded and deposited to an adjacent address. A repeated series of cycles with multiple jets enables rapid microarray production.

15. This makes use of semiconductor technologies. This ‘in situ’ fabrication technique was developed by Affymetrix , and is used to produce their Gene Chips . A mercury lamp is used , activates DNA bases. Photolithography

16. The photolithographic method Treat substrate with chemically protected “linker” molecules, creating rectangular array. Selectively expose array sites to light deprotects exposed molecules, activating further synthesis . Flush chip surface with solution of protected A,C,G,T. Binding occurs at previously deprotected sites. Repeat steps 2&3 until desired probes are synthesized.

17. The mask only allows light to pass to specific features on the chip.

18. A chip modified with photolabile protecting groups is selectively activated for DNA synthesis by shining light through a photomask . The chip is then flooded with a photoprotected DNA base, resulting in spatially defined coupling on the chip surface. A second photomask is used to deprotect defined regions of the chip . Repeated deprotection and coupling cycles enable the preparation of high- density oligonucleotide microarrays.

19. Principle of microarray technique Preparation of microarray Microspotting technique Piezoelectric printing Photolithography

20. DNA Microarray Technology High-throughput and versatile technology used for parallel gene expression analysis for thousands of genes of known and unknown functions. Used for detection of polymorphisms and mutations in genomic DNA A DNA microarray is a collection of microscopic DNA spots on solid surface. Each spot contains picomoles of a specific DNA sequence, known as probes or reporters .

21. Each identified sequenced gene on the glass, silicon chips or nylon membrane corresponds to a fragment of genomic DNA, cDNAs , PCR products or chemically synthesized oligonucleotides and represents a single gene. Probe-target hybridization is usually detected and quantified by detection of fluorophore , silver, or chemiluminescence labelled targets to determine relative abundance of nucleic acid sequences in the target.

22. The principle of DNA microarray technology is based on the fact that complementary sequences of DNA can be used to hybridise , immobilised DNA molecules.

23. Sample preparation Cy 3 Cy 5

24. Array Hybridisation

25. Slide is dried and scanned to determine how much labelled cDNA (probe) is bound to each target spot. Hybridized target produces emissions.

26. Upregulated genes Downregulated genes Equal abundance

27. Types of DNA Microarray/ DNA Chips cDNA based microarray Oligonucleotide based microarray

28. cDNA based microarray The first type of DNA microarray technology developed. This type of chips are prepared by using cDNA, it is called cDNA chips or cDNA microarray or probe DNA. The cDNAs are amplified by using PCR. These are immobilized on a solid support made up of nylon filtre of glass slide.

29. It was pioneered by Patrick Brown and his colleagues at Stanford University. Produced by using a robotic device which deposits (spots) a nanoliter of DNA onto a coated microscopic glass slide (50-150 µm in diameter).

30. Sample Preparation mRNA has been extracted from the cells or tissues under study, it is converted into DNA by the use of the reverse transcriptase enzyme. During this reaction, the DNA is labelled by the incorporation of fluorescent or radioactive nucleotides into the DNA. The two samples are labelled using two different fluorescent dyes - say, red or green . The most common dyes in use are Cy3 (Green) and Cy5 (Red) .

31. Oligonucleotide based microarray Often referred to as a "chip" which involves in situ oligonucleotide synthesis. Gene chip (DNA chip, Affymetrix chip) Oligonucleotide (20~80-mer oligos ) is synthesized in situ (on-chip). Developed at Affymetrix, Inc. , under the GeneChip ® trademark

32. AFFYMETRIX MICROARRAY

33. Affymetrix Chip Each gene has 16 – 20 pairs of probes synthesized on the chip. Each pairs of probes have two oligonucleotide . Perfect match (PM, reference seq ) ATG…C…TGC (20-25 bases). Mismatch (MM, one base change) ATG… T …TGC A MM oligo is identical to a PM oligo except that the middle nucleotide (13 th of 25) is intentionally replaced by its complementary nucleotide . The scanned result for a given gene is the average differences between PM and MM signals, over probes.

34. A Probe Set for Measuring Expression Level of a Particular Gene probe pair gene sequence

35. The black features represent no intensity (no RNA hybridized to the respective probe in the feature). The intensity level from lowest to highest by colour is: Dark blue -> Blue -> Light Blue -> Green -> Yellow -> Orange -> Red - > White . More intensity means more RNA bound to a specific feature, which basically means the gene was expressed at a higher level.

36. Affymetrix GeneChip experiment

37. Affymetrix GeneChip experiment Labelled cRNA randomly fragmented in to pieces anywhere from 30 to 400 base pairs in length. The fragmented, Biotin-labeled cRNA is added to the array Anywhere on the array where a cRNA fragment and a probe are complimentary, the cRNA hybridizes to the probes in the feature. The array is then washed to remove any cRNA that is not stuck to an array then stained with the fluorescent molecule that sticks to Biotin (Cy5 conjugated to streptavidin ). Lastly, the entire array is scanned with a laser and the information is kept in a computer for quantitative analysis of what genes were expressed and at what approximate level.

38. Procedure

39. Analysis of DNA microarray Bioconductor , an open source and open development software project for the analysis of genomic data primarily based on the R programming language, contains a number of program packages for microarray data analyses and is arguably the most comprehensive resource for such applications. (Gentleman et al., 2004)

40. Advantages of DNA microarray To study the behaviour of many genes simultaneously. The technique is very fast: there can be as many as 150 copies of an array of 12,000 genes printed in only 1 day. DNA microarray technology is relatively cheap to use: the initial cost of constructing an arrayer is approximately $60,000; after this, the cost per copy of a microarray is small, usually less than $100.

41. The technique of DNA microarrays is very user-friendly: the technique is neither radioactive nor toxic the microscope slide is a convenient base for the technique arrays are cheap and easily replaced A major advantage of DNA microarrays is that information about the sequence of the DNA is not required to construct and use the DNA microarrays.

42. Limitations As well as the cost of robotics to perform the technique, there may be technical limitations. The technique of DNA microarrays is currently limited not by the number of probes on an array, but by the resolution of the scanner used. Too much data all at once. Can take quite a while to analyze all the results. The results may be too complex to interpret The results are not always reproducible The results are not always quantitative enough The technology is still too expensive

43. Microarray in Genomics

44. The main application of genomic microarrays is represented by gene expression profiling . Basically, two types of genomic microarrays are available: wide genome and focused arrays . Wide-genome arrays are designed to bear on them as many genes as possible. Currently Affymetrix HU133 plus v.2 gene chips have around 47 000 genes or (ESTs) on them. Focused arrays are designed to bear few tens/hundreds of genes of interest.

45. Gene expression studies : data sets can vary greatly in size. The data heterogeneity is where the big challenge lies for the scientist, and a crucial role is thus played by bioinformatics and biostatistics. Softwares : Microarray Suite, GeneSpring , Partek Pro . All software packages are equipped with visualization tools, such as self-organizing maps, hierarchical clustering, principal component analysis , relevance networks, etc.

46. Self-organizing maps : genes are plotted on a two-dimensional graph based on their expression level across all samples. Thus, groups of genes with a similar expression pattern will have a similar trend within the graph.

47. Hierarchical clustering analyses: Genes are plotted against samples with a dendrogram , which is sort of a mock phylogenetic tree, whose branches connect genes related by a similar expression pattern: the shorter the branch, the stronger the correlation. The dendrogram is connected with its branches to the clustering map, where genes are represented by squares colored in green/red or blue/yellow, based on their differential expression level (up- or down-regulation), which define specific molecular fingerprints.

48. Even though visualization tools can give a comprehensive overview of the whole study performed, they are usually derived from the application of filters resulting in lists of at least hundreds of genes that might be difficult to interpret and make a sense of, without additional statistical analyses. Once a specific set of genes is identified as the significant one, validation studies are required in order to confirm gene expression profile observations.

49. Such validation should be preferentially done by RT-PCR, quantitative PCR ( Taqman ), Northern and Western blotting, RNA protection assay, flow cytometry , mice knockout models. All targeted to reconfirm previous gene expression profiling data.

50. cDNA microarrays containing ~9,000 unigenes was used to identify 486 salt responsive expressed sequence tags(ESTs) (representing ~450 unigenes ) in shoots of the highly salt-tolerant rice variety, Nona Bokra ( Oryza sativa L. ssp. Indica pv . Nona).

51. This study identified a large number of candidate functional genes that appear to be involved in salt tolerance and further examination of these genes may enable the molecular basis of salt tolerance to be elucidated. The rice BiostarP-100s cDNA microarray (United Gene Holdings,Ltd ., PRC), containing 10,060 sequences representing ~9000 unigenes including novel, known and control genes, was used to identify salt-regulated genes.

52. Gene expression was examined at three time points after salt treatment (20min, 3h and 24h) corresponding to early transient, intermediate and late regulation. The significantly regulated genes at each time point were selected for cluster analysis and for inclusion in the salt-induced-microarray (SIM).

53. The fluorescent signatures were scanned and captured using a ScanArray4000 Standard Biochip Scanning System. Data were analyzed using the GenePix Pro 3.0 software.

54. The utilized genes were amplified by polymerase chain reaction (PCR) of the appropriate rice cDNA clones using T3 and T7 primers. After the resulting products were purified and confirmed by direct sequencing, the fragments were printed on slides using an OmniGrid printer.

55. Red : up-regulated genes Green: down-regulated genes; Black: un-regulated genes Blanks: missing data. Comparison of salt response gene expression in salt-stressed Nona and IR28 plants using hierarchical cluster analysis

56. Advantages of genomic microarray One shot genome wide expression analysis. Rapid comparison between two states (control/diseased, untretead /treated, and wild type/knockout). Exploration of new biological systems in a hypotheses generating rather than hypotheses testing fashion. Identification of markers to elucidate molecular mechanisms (signatures) underlying biological events and diseases. Rapid molecular disease classification for more accurate molecular diagnostic, prognostic and targeted treatment.

57. Disadvantages Restricted access to the technology (experiments still exprehensive to perform) Not yet approved as diagnostic tool by regulatory bodies Not a stand alone technique (need validation/confirmation tests) Skilled technical personnel needed (including biostatistician/ bioinformatician for data analysis) Data derived from different platforms difficult to compare Data comparability difficult from one array version to the next Data obtained only partially used and published Data repositories and data sharing still not fully implemented Ethical and legal issues when dealing with patient samples

58. Protein microarrays are miniaturized and parallelized array technology approaches for protein–protein interactions analysis and protein profiling. Typically, thousands of proteins are printed and immobilized on functionalized glass slides, which that can be simultaneously studied and analyzed in a HT fashion, thereby offering a high potential for characterizing the biology of a given cell of interest. PROTEIN MICROARRAY

59. The first report of using protein arrays for protein–protein interaction, ligand binding and biochemical investigations was by MacBeath and Schreiber in 2000 . The success of each microarray-based screening heavily depends on the library construction and microarray fabrication. Three key areas of protein characterizations : functional annotation, substrate fingerprinting, ligand /inhibitor binding.

60. The major challenge as compared to DNA microarray is that the need to maintain the structural integrity and physicochemical properties of proteins, derived from its complexity & variability (e.g. post-translational modifications, etc.).

61. Classification of Protein Microarray Target microarrays, Reverse Phase arrays in situ expressed arrays.  

62. Detection techniques for Protein microarray Conventional fluorescence labeling : the use cyanine dyes (e.g.: Cy3 and Cy5) Target protein array NAPPA protein array ( Nucleic Acids Programmable Protein Arrays ) Reverse phase microarray Detection for suspension array technology Flow cytometry Magnetic bead based detection Quantum dots Gold Nano particles Label free techniques Surface Plasmon Resonance Microcantilevers and Atomic force microscopy