DNA Microarray Steps (Procedure) and Applications

DNA Microarray Definition and Principle              
DNA Microarray Procedure                                   
1) Collect Samples: This can be from a variety of organisms. Two samples : cancerous human skin tissue & healthy human skin tissue

2) Isolate mRNA:

• Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.
• After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind the mRNA.
• Rinse with buffer to release the mRNA from the beads. The buffer disrupts the pH, disrupting the hybrid bonds.

3) Create labelled cDNA:

• Add a labelling mix to the RNA. The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.
• We will add cyanine 3  (cy3-fluoresces green) to the healthy cells and cyanine 5 (cy5-fluoresces red) to the cancerous cells.
• The primer and RT bind to the mRNA first, then add the fluorescently dyed nucleotides, creating a complementary strand of DNA

4) Hybridization

• Apply the cDNA we have just created to a microarray plate.
• When comparing two samples, apply both samples to the same plate.
• The ssDNA will bind to the cDNA already present on the plate.

5)Detect the relative intensities of fluorescence under Microarray Scanner

• The scanner has a laser, a computer, and a camera.
• The laser causes the hybrid bonds to fluoresce.
• The camera records the images produced when the laser scans the plate.
• The computer allows us to immediately view our results and it also stores our data.

6) Analyze Data

Application of DNA microarray

• It is used in the analysis  of transcriptomes and proteomes.
• Gene chips are available to diagnose several pathogenic and genetic diseases in man.
• With the help of species specific probes, DNA microarray is used to identify microbes in the environment.
• It is employed in genotyping of genomes through single nucleotide polymorphism (SNP) analysis.
• DNA microarray is used to detect gene expression by analyzing cDNAs produced from mRNAs of a cell type at different times.
• To measure changes in gene expression levels – two samples’ gene expression can be compared from different samples, such as from cells of different stages of mitosis.
• To observe genomic gains and losses. Microarray Comparative Genomic Hybridization (CGH)
• To observe mutations in DNA.