Microarray FAQ

The following is meant to help people understand what DNA microarrays are, how they are made, and why they are useful. This is a work in progress, if you have any corrections, clarifications or a new question don't hesitate to email me at sugnet@cse.ucsc.edu.

The Questions:
y y y y y y y y y What is a microarray? how are microarrays made? So what are the differences between different types of microarrays? how are microarrays used? Why ratios and cohybridizations? What is a "hybridization"?, how does it work? What is "Reverse Transcription"? how sensitive are microarrays? I still have lots of questions, where can I find more information?

The Answers:
What is a microarray?
A generic microarray consists of multiple features (spots) of DNA which will be used to determine the levels of mRNA expression in a collection of cells. The DNA for each feature is from a gene of interest and is a probe for the mRNA encoded by that gene. In general you can think of a microarray as a grid of DNA spots. Each spot has a unique DNA sequence, different from the DNA sequence of the other spots around it. Thus each spot will hybridize only to its complementary DNA strand. In this way each spot is acting as a probe to determine the levels of a specific mRNA produced by a collection of cells. The basic idea of using a piece of DNA as a probe to determine the presence of the complementary DNA in a solution goes back a long time. It is the same general technique used in Southern, and Northern blots by molecular biologists every day. The thing that makes microarrays so exciting now is the number of DNA probes that it is possible to place on a microarray. Already there are microarrays with probes for every gene in Yeast, and others with over 19,000 human cDNAs. This allows researchers to observe the response of whole genomes to various stimuli instead of one gene at a time.

To the left is an image of a portion of a microarray that has been hybridized and scanned. Each spot gives information as to the relative abundance of the mRNA which is complementary to the DNA in that spot. In this manner it is possible to gather mRNA expression levels for thousands of genes at the same time in parallel.

how are microarrays made?

Currently microarrays come in many different types but they are only two real fabrication methods. 1. Synthesize DNA probes separately, using PCR for cDNAs or chemical synthesis for oligonuclotides. Then use a robot to spot these DNA probes onto your microarrays into very small grids. The substrate for your microarrays can be glass, plastic, or even nylon membranes. Most labs I know of are using glass microscope slides. This method is compartively cheap and flexible and Pat Brown's lab posted plans to make the robot on the web. Some related technologies use an ink-jet like printer to spray oligonucleotide probes on the microarrays. 2. Synthesize DNA oligonucleotides directly on the microarray using UV-masks and photo-activated chemistry. Currently Affymetrix are the only people doing this. Affymetrix is a dominant player in commercial microarrays and the chemistry they use is just amazing. The technique used is as follows: deprotect sites that will have the next base (A,C,T, or G) bound to them using UV light, then bind the next base to those sites and repeat with a different base. To direct which sites will be deprotected Affymetrix uses a photolithographic mask which only lets the UV light activate certain sites.

Using this technology Affymetrix is able to build up very large arrays of oligonucleotides in parallel. however due to synthesis efficiencies the longest oligonucleotide probes that Affymetrix makes are 25 nucleotides long.

So what are the differences between different types of microarrays?

The different types of microarrays each have their own peculiarities and no one that I know of has published any sort of study rigorously comparing the different technologies. however there are some inherent strengths and weaknesses to each technology. y The Affymetrix chemistry is great, but their technology is very expensive and fairly inflexible. The basic Fluidics station and scanner are over $100,000 and then each GeneChip is around $5,000. The reason that it is inflexible is that if you don't like their arrays and want to make your own the cost for a new photolithographic mask can be over a million dollars. That said, the technology is very robust and Affymetrix's chemistry is reproducible and allows the detection of SNPs and other small features in the DNA. One thing to note is that Affymetrix is not currently doing cohybridizations which makes it very important and challenging to normalize between the experimental and control GeneChips. That is to say that Affymetrix does not produce ratios, each probe produces only an absolute intensity. y Spotting DNA on glass microscope slides is relatively inexpensive and very flexible. however the spotting process itself is inherently variable. Also most microarrays produced in this manner use cDNAs as their probes. Using cDNAs has a couple of techinical problems.

1. You need a copy of that DNA to start with so you can use PCR to produce your probes. This is a major point as we know the sequence of whole genomes but we don't have unique cDNA libraries that span genomes. 2. When using a cDNA as a probe you get a very long sequence to bind to which makes it impossible to discern between genes that are more than 80% similar, and forget about detecting SNPs. It is possible to spot oligos on glass slides and save yourself a lot of PCR and avoid the above limitations. The technique that allows the spotting technology to sidestep the issue of variability in spotting and other concerns is the use of cohybridizations. This technique is covered in greater detail later in this document but the main concept is to use relative RNA expression levels instead of absolute expression levels. To accomplish this two separate RNA samples are used: an "experimental" and a "reference". Each RNA is labeled with a different fluorescent dye, then the two samples are mixed and hybridized at the same time to the microarray. When the microarray is scanned, number of photons in the experimental dye's spectrum is compared to the number of photons in the reference dye's spectrum. Many variations in spot size, probe concentration and other issues are cancelled out in this manner.

how are microarrays used?

The basic protocol is as follows: 1. Isolate the RNA you are interested in and the RNA from your control. The RNA can come from any cells. It is important to realize though that the RNA from tissues or any heterogenous cells may lead to results that reflect changes in the composition of the sample rather than in changes due to the experimental hypothesis. 2. Label the RNA. Usually this means preforming a reverse transcriptase reaction and incorporating dye that has been linked to a DNA nucleotide. however some protocols, i.e. Affymetrix's, call for an amplification of the RNA and labelling of the RNA itself. For microarrays on nylon membranes usually the label is radioactive. 3. hybridize the labeled target to the microarray. This consists of placing a solution containing the labeled target on the microarry and letting it sit for a period of hours. This allows a given target to find it's probe on the microarray and bind to it. Usually this is carried out a specific temperature to minimize non-specific binding of target to the probes on the microarray. 4. Remove the hybridization solution and wash the microarray. The washing can be done at different salt and detergent concentrations to minimize non-specific binding. In general solutions with lower salt concentrations weaken the DNA base paring and are referred to as "more stringent" and vice versa for higher salt concentrations. 5. Once the microarray has been washed it is time to scan the microarray. Scanning is just quantitizing how much target bound to the DNA probe on the microarray. Most microarrays use fluorescent dyes and are scanned in the following manner: 1. laser is used to excite the fluorescent dye, the photons coming from the dye are captured using lenses to focus the light and a photo multiplier tube (PMT) to quanitate how many photons are being captured. 2. The resulting number for that section of the microaray is translated into one pixel of a 16 bit .tiff file. The more pixels per centimeter, the better the resolution of the resulting .tiff

image. It is important to note that .tiff files are uncompressed and file formats like .jpeg and .gif which compress data should not be used for storage of results. 3. The resulting image is analyzed by finding the spots and comparing the differences between chips (if the hybridization contained only one fluor) or the ratio of the two fluors for cohybridization experiments. how these differences are normalized, compared and interpreted is beyond the scope or this document.

For more information on scanning basics check out Axon's FAQ.

Why ratios and cohybridization?

It can be difficult using microarrays to show that there is a relationship between the absolute intensity of a hybridized probe and the absolute number of target molecules bound. This is especially true with cDNA probes where it may be difficult to control what sequences are present in a probe. Issues such as secondary structure, melting temperature, and even target characteristics make it hard to calibrate an entire array for measuring absolute molecules bound. In order to sidestep these issues researchers use cohybridizations to measure the level of RNA expression relative to another sample at the same time on the same microarray. The basic idea is as follows: 1. Isolate the RNA from the experimental state and label it with a fluorescent dye, usually Cy5 or Cy3. 2. Isolate RNA from a control state (also called a reference state) and label it with a different fluorescent dye, usually Cye3 or Cy5, whichever you didn't use for the experimental RNA. 3. Mix the two samples and hybridize them at the same time on the same microarray. 4. Wash the microarry to remove non-specific binding and scan the microarray to quantitate the amount of each fluor, both the control and the experimental. The key idea is now to not analyze the absolute intensities, but rather to compare how different the experimental is from the control. This is usually expressed as a ratio of the two numbers. There is currently much debate about how to use and normalize these ratios, and even exactly which numbers to use for the ratios. The actual number used to do the ratios comes from the intensities of the pixels that make up the spot of that DNA probe. Once these pixel intensities are adjusted for background there are many ways to extract a single number to use in ratios. Some people use the median pixel value, some use the mean, some researches throw out all of the saturated pixels and then take the mean. I highly reccomend finding a good statistics book and familiarizing yourself with basic statistics agian to understand the significance of these numbers and how they effect your results. Once you've decided how you want to extract a single value from the pixel intensities you can now measure your ratio and determine if your genes of interest are being expressed more or less relative to your control sample. Some researchers take the log of these ratios as they will then have the nice property of being centered around zero with positive numbers indicating induction a gene and negative numbers indicating repression a gene, relative to the control sample. Keep in mind that there are still a host of issues when it comes to normalizing these values and comparing values between different microarrays. These issues are outside the scope of this document.

What is a "hybridization"?
In order to understand how hybridizations work check out my Biology starter to see the how this technique exploits the marvelous properties of DNA.

What is "Reverse Transcription"?

Reverse transcrition uses and enzyme conveniently named Reverse Transcriptase to produce the complementary DNA strand from an RNA strand. To find out more about the amazing properties of DNA and RNA check out my Biology starter

how sensitive are microarrays?

Affymetrix claims that on a routine basis they can detect 1 molecule in 100,000 and that they can detect two fold changes in RNA expression levels. In an optimal hybridization they claim to detect one molecule in 2,000,000 and to detect 10% changes in RNA expression levels. I haven't seen any published results on spotted arrays but I'd love to hear about it if someone else has.

I still have lots of questions, where can I find more information?

Check out other sources of information or emailme. The Mguide Build your own and do it yourself. The Brown Lab FAQ Specific technical questions. Axon's FAQ. Image scanning quesions. Check out the Microarray Links page for other resources.

Microarray Technology
How microarray technology works? Bacterial identification using microarrays Gene splicing detection using microarrays

Introduction to Microarray:
Molecular Biology research evolves through the development of the technologies used for carrying them out. It is not possible to research on a large number of genes using traditional methods. DNA Microarray is one such technology which enables the researchers to investigate and address issues which were once thought to be non traceable. One can analyze the expression of many genes in a single reaction quickly and in an efficient manner. DNA Microarray technology has empowered the scientific community to understand the fundamental aspects underlining the growth and development of life as well as to explore the genetic causes of anomalies occurring in the functioning of the human body. A typical microarray experiment involves the hybridization of an mRNA molecule to the the DNA template from which it is originated. Many DNA samples are used to construct an array. The amount of mRNA bound to each site on the array indicates the expression level of the various genes. This number may run in thousands. All the data is collected and a profile is generated for gene expression in the cell.

Microarray Technique
An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. The sample spot sizes are typically less than 200 microns in diameter usually contain thousands of spots. Thousands of spotted samples known as probes (with known identity) are immobilized on a solid support (a microscope glass slides or silicon chips or nylon membrane). The spots can be DNA, cDNA, or oligonucleotides. These are used to determine complementary binding of the unknown sequences thus allowing parallel analysis for gene expression and gene discovery. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. An orderly arrangement of the probes on the support is important as the location of each spot on the array is used for the identification of a gene.

Types of Microarrays:
Depending upon the kind of immobilized sample used construct arrays and the information fetched, the Microarray experiments can be categorized in three ways: 1. Microarray expression analysis: In this experimental setup, the cDNA derived from the mRNA of known genes is immobilized. The sample has genes from both the normal as well as the diseased tissues. Spots with more more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease. 2. Microarray for mutation analysis: For this analysis, the researchers use gDNA.

The genes might differ from each other by as less as a single nucleotide base. A single base difference between two sequences is known as Single Nucleotide Polymorphism (SNP) and detecting them is known as SNP detection 3. Comparative Genomic Hybridization: It is used for the identification in the increase or decrease of the important chromosomal fragments harboring genes involved in a disease.

Applications of Microarrays: Gene discovery: DNA Microarray technology helps in the identification of new genes, know
about their functioning and expression levels under different conditions.

Disease diagnosis: DNA Microarray technology helps researchers learn more about different
diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.

Drug discovery: Microarray technology has extensive application in Pharmacogenomics.Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect. Toxicological research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. GEO:
In the recent past, microarray technology has been extensively used by the scientific community. Consequently, over the years, there has been a lot of generation of data related to gene expression. This data is scattered and is not easily available for public use. For easing the accessibility to this data, the National Center for Biotechnology Information (NCBI) has formulated the Gene Expression Omnibus or GEO. It is a data repository facility which includes data on gene expression from varied sources.

Microarray probe design parameters:

For 25-35 mers

Parameter Probe Length Probe Length tolerance Probe Target Tm Probe Tm Tolerance (+) Hairpin Max G Self Dimer G Run/Repeat

Minimum Value 10 0 40 0.1 0.1 0.1 2

Maximum Value 99 15 99 99 99.9 99.9 99

Default Value 30 3 63 5 4 7 4

Unit bases

Kcal/mol Kcal/mol bases

For 35-45 mers " Parameter Probe Length Probe Length tolerance Probe Target Tm Probe Tm Tolerance (+) Hairpin Max G Self Dimer G Run/Repeat For 65-75 mers Minimum Value 10 0 40 0.1 0.1 0.1 2 Maximum Value 99 15 99 99 99.9 99.9 99 Minimum Value 10 0 40 0.1 0.1 0.1 2 Maximum Value 99 15 99 99 99.9 99.9 99 Default Value 40 3 70 5 6 8 5 Kcal/mol Kcal/mol bases C Unit bases

Parameter Probe Length Probe Length tolerance Probe Target Tm Probe Tm Tolerance (+/above ) Hairpin Max G Self Dimer G Run/Repeat

Default Value 70 3 75 5 6 8 6

Unit bases

Kcal/mol Kcal/mol bases

Other Parameters

Probe Location 1.3' end bias: The oligos chosen should be towards the 3' end of the gene i.e. Default : 3' end. 2.The oligos should be designed by default within 999 bases of 3' end. The range can be from 0 to 1500 bases. The oligos should be free of cross homology (i.e They should be BLAST searched against the appropriate genome category).

DNA Microarray with Array Designer: Array Designer is an exceptional software to design highly specific oligos for expression and SNP genotyping microarray experiments.

References:
1.Validation of Sequence-Optimized 70-base Oligonucleotides for Use on DNA Microarrays (http://www.westburg.nl/download/arrayposter.pdf). By John Ten Bosch, Chris Seidel, Sajeev Batra, Hugh Lam, Nico Tuason, Sepp Saljoughi, and Robert Saul. 2. Assessment of the specificity and Sensitivity of the oligonucleotides (50 mer ) microarrays. By Dr. Susanne Schrder1, Dr. Jaqueline Weber2, and Dr. Hubert Paul 1 WG Biotech AG, Microarray Development1 and Department of Bioinformatics2. 3. 50 nucleotide long probes on microarrays enable high signal intensity and high specificity. By Dr. Susanne Schrder1, Dr. Jaqueline Weber2, and Dr. Hubert Paul1 MWG Biotech AG,microarray Development1and Department of Bioinformatics 2, Anzinger Str. 7, 85560 Ebersberg, Germany. 4.Optimization of Oligonucleotide - based DNA microarrays. By Angela Relogio, Christian Aschwager, Alexandra Ritcher, Wilhelm Ansorge and Juan Valcarel. DNA Microarray Technique

Single-nucleotide polymorphism
From Wikipedia, the free encyclopedia

DNA molecule 1 differs from DNA molecule 2 at a single base-pair location (a C/T polymorphism).

A single-nucleotide polymorphism (SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide A, T, C, or G in thegenome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles: C and T. Almost all common SNPs have only two alleles.

Within a population, SNPs can be assigned a minor allele frequency the lowest allele frequency at a locus that is observed in a particular population. This is simply the lesser of the two allele frequencies for single-nucleotide polymorphisms. There are variations between human populations, so a SNP allele that is common in one geographical or ethnic group may be much rarer in another.

Types of SNPs
Types of SNPs   Non-coding region Coding region   Synonymous Nonsynonymous   Missense Nonsense

Single nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. A SNP in which both forms lead to the same polypeptide sequence is termed synonymous (sometimes called a silent mutation) if a different polypeptide sequence is produced they are nonsynonymous. A nonsynonymous change may either be missense or nonsense, where a missense change results in a different amino acid, while a nonsense change results in a prematurestop codon. SNPs that are not in protein-coding regions may still have consequences for gene splicing, transcription factor binding, or the sequence of non-coding RNA. [edit]Use

and importance of SNPs

Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. SNPs are also thought to be key enablers in realizing the concept of personalized medicine.[1] However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as with matched cohorts with and without a disease). The study of single-nucleotide polymorphisms is also important in crop and livestock breeding programs (see genotyping). See SNP genotyping for details on the various methods used to identify SNPs.

They are usually biallelic and thus easily assayed.

[3]

[2]

SNPs do not necessarily function individually, rather,

they work in coordination with other SNPs to manifest a disease condition as has been seen in osteoporosis See

Examples
    rs6311 and rs6313 are SNPs in the HTR2A gene on human chromosome 13. A SNP in the F5 gene causes a hypercoagulability disorder with the variant Factor V Leiden. rs3091244 is an example of a triallelic SNP in the CRP gene on human chromosome 1. TAS2R38 codes for PTC tasting ability, and contains 6 annotated SNPs.
[5] [4]

[edit]Databases As there are for genes, there are also bioinformatics databases for SNPs. dbSNP is a SNP database from National Center for Biotechnology Information (NCBI). SNPedia is a wiki-style database from ahybrid organization. The OMIM database describes the association between polymorphisms and, e.g., diseases in text form, HGMD the Human Gene Mutation Database provides gene mutations causing or associated with human inherited diseases and functional SNPs, while HGVbaseG2P allows users to visually interrogate the actual summary-level association data. [edit]Nomenclature The nomenclature for SNPs can be confusing: several variations can exist for an individual SNP and consensus has not yet been achieved. One approach is to write SNPs with a prefix, period and greater than sign showing the wild-type and altered nucleotide or amino acid; for example, c.76A>T.[6][7][8]

SNP array
From Wikipedia, the free encyclopedia

In molecular biology and bioinformatics, a SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. For example, there are around 10 million SNPs that have been identified in the human genome[1]. As SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research.

Principles
The basic principles of SNP array are the same as the DNA microarray. These are the convergence of DNA hybridization, fluorescence microscopy, and solid surface DNA capture. The three mandatory components of the SNP arrays are:

1. The array that contains immobilized nucleic acid sequences or target; 2. One or more labeled allele-specific oligonucleotide (ASO) probes; 3. A detection system that records and interprets the hybridization signal. To achieve relative concentration independence and minimal cross-hybridization, raw sequences and SNPs of multiple databases are scanned to design the probes. Each SNP on the array is interrogated with different probes. Depending on the purpose of experiments, the amount of SNPs present on an array is considered.

Applications
An SNP array is a useful tool to study the whole genome. The most important application of SNP array is in determining disease susceptibility and consequently, in pharmacogenomics by measuring the efficacy of drug therapies specifically for the individual. As each individual has many single nucleotide polymorphisms that together create a unique DNA sequence, SNP-based genetic linkageanalysis could be performed to map disease loci, and hence determine disease susceptibility genes for an individual. The combination of SNP maps and high density SNP array allows the use of SNPs as the markers for Mendelian diseases with complex traits efficiently. For example, whole-genome genetic linkage analysis shows significant linkage for many diseases such as rheumatoid arthritis,prostate cancer, and neonatal diabetes. As a result, drugs can be personally designed to efficiently act on a group of individuals who share a common allele - or even a single individual. A SNP array can also be used to generate a virtual karyotype using specialized software to determine the copy number of each SNP on the array and then align the SNPs in chromosomal order. In addition, SNP array can be used for studying the Loss of heterozygosity (LOH). LOH is a form of allelic imbalance that can result from the complete loss of an allele or from an increase in copy number of one allele relative to the other. While other chip-based methods (e.g. Comparative genomic hybridization) can detect only genomic gains or deletions, SNP array has the additional advantage of detecting copy number neutral LOH due to uniparental disomy (UPD). In UPD, one allele or whole chromosome from one parent are missing leading to reduplication of the other parental allele (uni-parental = from one parent, disomy = duplicated). In a disease setting this occurrence may be pathologic when the wildtype allelle (e.g. from the mother) is missing and instead two copies of the mutant allelle (e.g. from the father) are present. Using high density SNP array to detect LOH allows identification of pattern of allelic imbalance with potential prognostic and diagnostic utilities. This usage of SNP array has a huge potential in cancer diagnostics as LOH is a prominent characteristic of most human cancers. Recent studies based on the SNP array technology have shown that not only solid tumors (e.g. gastric cancer, liver cancer etc) but also hematologic malignancies (ALL, MDS, CML etc) have a high rate of LOH due to

genomic deletions or UPD and genomic gains. The results of these studies may help to gain insights into mechanisms of these diseases and to create targeted drugs.