outline how microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression

Principles of Genetic Technology

Microarrays: Overview

Microarrays are solid‑phase platforms that contain thousands of DNA probes arranged in a precise grid. They enable simultaneous interrogation of many genetic sequences, making them powerful tools for both genome analysis and the detection of messenger RNA (mRNA) in gene‑expression studies.

How a Microarray Works

1. Probe Design and Spotting – Short DNA fragments (probes) are immobilised on a glass slide, silicon chip or nylon membrane at defined coordinates.
2. Sample Preparation – Genomic DNA or cDNA derived from mRNA is extracted and labelled with a fluorescent dye (e.g., Cy3 or Cy5).
3. Hybridisation – The labelled sample is applied to the array and allowed to hybridise to complementary probes. The reaction can be represented as:

   \$\text{Probe} + \text{Labelled target} \rightleftharpoons \text{Probe–target duplex}\$

4. Washing – Non‑specific bindings are removed, leaving only stable duplexes.
5. Scanning – A laser scanner excites the fluorophores; emitted light intensity at each spot is recorded.
6. Data Analysis – Fluorescence intensity is quantified, normalised and interpreted using bio‑informatic tools.

Types of Microarrays

Applications in Genome Analysis

• Single Nucleotide Polymorphism (SNP) Genotyping – Oligonucleotide arrays contain probes for known SNP loci; hybridisation patterns reveal genotype.
• Comparative Genomic Hybridisation (CGH) – Test and reference genomes are differentially labelled and co‑hybridised; fluorescence ratios indicate gains or losses of chromosomal regions.
• Copy‑Number \cdot ariation (CNV) Mapping – High‑resolution arrays detect sub‑microscopic deletions or duplications linked to disease.

Detecting mRNA in Gene‑Expression Studies

When studying gene expression, the target material is cDNA generated from cellular mRNA. The steps are adapted as follows:

1. Isolate total RNA from the tissue or cells of interest.
2. Reverse‑transcribe RNA to cDNA, incorporating fluorescent nucleotides.
3. Hybridise labelled cDNA to a gene‑specific array (often an oligonucleotide or cDNA array).
4. Scan and quantify fluorescence; intensity at each spot reflects the abundance of the corresponding mRNA in the original sample.

Comparative experiments (e.g., treated vs. control) use two different dyes (Cy3 and Cy5) on the same array, allowing direct ratio calculations for up‑ or down‑regulation.

Data Interpretation

Raw fluorescence values are processed through several steps:

• Background Subtraction – Removes non‑specific signal.
• Normalization – Adjusts for dye bias and variation between arrays (e.g., using the median or loess method).
• Statistical Analysis – Determines which genes show significant expression changes (t‑test, ANO \cdot A, false‑discovery‑rate correction).

Advantages and Limitations

Key Points to Remember

• Microarrays rely on the principle of complementary base‑pairing between probes and labelled targets.
• Fluorescent labelling enables detection of hybridisation events by scanning.
• They are versatile: used for SNP genotyping, CGH, and quantitative gene‑expression profiling.
• Data must be carefully normalised and statistically evaluated to draw reliable biological conclusions.