DNA microarray experiment
Encyclopedia
- The two samples to be compared (pairwise comparison) are grown/acquired. In this example treated sample (caseCase-controlA case-control study is a type of study design in epidemiology. Case-control studies are used to identify factors that may contribute to a medical condition by comparing subjects who have that condition with patients who do not have the condition but are otherwise similar .Case-control studies are...
) and untreated sample (controlCase-controlA case-control study is a type of study design in epidemiology. Case-control studies are used to identify factors that may contribute to a medical condition by comparing subjects who have that condition with patients who do not have the condition but are otherwise similar .Case-control studies are...
). - The nucleic acidNucleic acidNucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
of interest is purified: this can be all RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
for expression profilingExpression profilingIn the field of molecular biology, gene expression profiling is the measurement of the activity of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between cells that are actively dividing, or show how the cells react to a...
, DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
for comparative hybridization, or DNA/RNA bound to a particular proteinProteinProteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
which is immunoprecipitated (ChIP-on-chipChIP-on-chipChIP-on-chip is a technique that combines chromatin immunoprecipitation with microarray technology . Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo...
) for epigeneticEpigeneticsIn biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- -genetics...
or regulation studies. In this example total RNA is isolated (total as it is nuclear and cytoplasmCytoplasmThe cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
ic) by Guanidinium thiocyanate-phenol-chloroform extraction (e.g. TrizolTrizolTRIzol is a chemical solution used in RNA/DNA/protein extraction, by the reference paper from Piotr Chomczyński and Sacchi, N. in 1987.TRIzol is the brand name of the product from Invitrogen, and the brand name from MRC, which was founded by Chomczynski, is TRI Reagent.The correct name of the...
) which isolates most RNA (whereas column methods have a cut off of 200 nucleotides) and if done correctly has a better purity. - The purified RNA is analysed for quality (by capillary electrophoresisCapillary electrophoresisCapillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...
) and quantity (by using a spectrophotometer like NanoPhotometerTM or nanodropSpectrophotometryIn chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength...
): if enough material (>1μg) is present the experiment can continue. - The labelled product is generated via reverse transcription and sometimes with an optional PCR amplification. The RNA is reverse transcribed with either polyT primers which amplify only mRNA or random primers which amplify all RNA which is mostly rRNA, miRNA microarray ligate an oligonucleotide to the purified small RNA (isolated with a fractionator) and then RT and amplified. The label is added either in the RT step or in an additional step after amplification if present. The sense that is labelled depends on the microarray, which means that if the label is added with the RT mix, the cDNA is on the template strand while the probe is on the sense strand (unless they are negative controls). The label is typically fluorescent; only one machine uses radiolabels. The labelling can be direct (not used) or indirect which requires a coupling stage. The coupling stage can occur before hybridization (two-channel arrays) using aminoallyl-UTP and NHSN-HydroxysuccinimideN-Hydroxysuccinimide is a compound with a molecular weight of 115.09 and a melting point of 95 °C.As it is slightly acidic, it is an irritant to skin, eyes and mucous membranes....
amino-reactive dyes (like cyanine dyesCyanineCyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...
) or after (single-channel arrays) using biotin and labelled streptavin. The modified nucleotides (typically a 1 aaUTP: 4 TTP mix) are added enzymatically at a lower rate compared to normal nucleotides, typically resulting in 1 every 60 bases. The aaDNA is then purified with a columnDNA separation by silica adsorptionDNA Separation by Silica Adsorption is an important method of DNA separation that is used in novel technologies that use micro-channels. The principle behind this type of separation relies on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH...
(using solution containing phosphate buffer as Tris contains amine groups). After purification, labelling efficiency (Frequency of Incorporation (FOI)CyanineCyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...
) of each sample has to be measured with a small volume photometer (like the NanoPhotometerTM) to ensure equal amounts of dye incorporation to the samples to be compared. The aminoallyl group is an amine group on a long linker attached to the nucleobase, which reacts with a reactive dye. A dye flip is a type of replicate done to remove any dye effects in two-channel dyes, in one slide one same is labeled with Cy3 the other with Cy5, this is reversed in a different slide. In this example, in the presence of aminoallyl-UTP added in the RT mix. - The labeled samples are then mixed with a propriety hybridization solution which may contain SDSSodium dodecyl sulfateSodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...
, SSCCitrateA citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid. An example of the former, a salt is trisodium citrate; an ester is triethyl citrate.-Other citric acid ions:...
, dextran sulfateDextranDextran is a complex, branched glucan composed of chains of varying lengths...
, a blocking agent (such as COT1 DNA, salmon sperm DNA, calf thymus DNA, PolyA or PolyT), Denhardt's solution and formamineMethylamineMethylamine is the organic compound with a formula of CH3NH2. This colourless gas is a derivative of ammonia, but with one H atom replaced by a methyl group. It is the simplest primary amine. It is sold as a solution in methanol, ethanol, THF, and water, or as the anhydrous gas in pressurized...
. - This mix is denatured and added to a pin hole in a microarray, which can be a gene chip (holes in the back) or a glass microarray which is bound by a cover, called a mixer, containing two pinholes and sealed with the slide at the perimeter.
- The holes are sealed and the microarray hybridized, either in a hyb oven, where the microarray is mixed by rotation, or in a mixer, where the microarray is mixed by alternating pressure at the pinholes.
- After an overnight hybridization, all nonspecific binding is washed off (SDS and SSC).
- The microarray is dried and scanned in a special machine where a laser excites the dye and a detector measures its emission.
- The image is gridded with a template and the intensities of the features (several pixels make a feature) are quantified.
- The raw data is normalized, the simplest way is to subtract the background intensity and then divide the intensities making either the total intensity of the features on each channel equal or the intensities of a reference gene and then the t-value for all the intensities is calculated. More sophisticated methods include z-ratio, loess and lowess regressionLocal regressionLOESS, or LOWESS , is one of many "modern" modeling methods that build on "classical" methods, such as linear and nonlinear least squares regression. Modern regression methods are designed to address situations in which the classical procedures do not perform well or cannot be effectively applied...
and RMA (robust multichip analysis) for Affymetrix chips (single-channel, silicon chip, in situ synthesised short oligonucleotides).
See also
- DNA microarrayDNA microarrayA DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
- MicrofluidicsMicrofluidicsMicrofluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
or lab-on-chip - Quantification of nucleic acidsQuantification of nucleic acidsIn molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and purity for optimum performance...
- CyanineCyanineCyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...
dyes, such as Cy3 and Cy5 - Protocol (natural sciences)Protocol (natural sciences)In the natural sciences a protocol is a predefined written procedural method in the design and implementation of experiments. Protocols are written whenever it is desirable to standardize a laboratory method to ensure successful replication of results by others in the same laboratory or by other...
- sample replicationReplicationReplication may refer to:Science* Replication is one of the main principles of the scientific method, a.k.a. reproducibility** Replication , the repetition of a test or complete experiment...
- Molecular biologyMolecular biologyMolecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
- Serial analysis of gene expressionSerial Analysis of Gene ExpressionSerial analysis of gene expression is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu...