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Microarray Experiment Overview

This repository documents a microarray experiment conducted on a glass slide as a medium for gene expression analysis. The microarray slides contain spots filled with short oligonucleotide sequences (probes) that are complementary to the cDNA of the sample.

Glass Slide Construction

  • Company: Affymetrix manufactures the glass slides.
  • Spots: The spots on the glass slide contain short oligonucleotide probes.
  • Purpose: These probes are used to measure gene expression levels in a sample.

Signal Value

The signal value in a microarray experiment indicates the level of gene expression:

  • Higher signal value → Higher fluorescence → Higher gene expression

Techniques to Measure Gene Expression

1. Polymerase Chain Reaction (PCR)

  • Measures the expression of one or a few genes at a time.
  • Primers: Short oligonucleotide sequences (18-23 bp).

2. Microarray

  • Measures the expression of thousands of genes simultaneously.
  • Probes: 40-70 bp in length.
  • Requires prior information about the genes being studied.

3. Next-Generation Sequencing (NGS)

  • Baits: Up to 120 bp in length.
  • Used for targeted resequencing.

All primers, baits, and probes in these techniques are designed using the Tiling algorithm.

Array Format

The choice of array format depends on the number of samples and features. For example:

  • 8*15K Format:
    • 8 samples.
    • Each sample contains 15,000 features (spots).
    • Empty spots can be filled with probes of replicates.

Steps of a Microarray Experiment

1. Probe Designing (Bioinformatics)

  • Designing probes is the first step and involves bioinformatics.
  • The process takes 3-4 months to complete.
  • Steps of Probe Designing

2. Hybridization (Wet Lab Technique)

  • Control: Cy3 (Green)
  • Treated: Cy5 (Red)
  • In this process, labeled cDNA binds to the probes on the glass slide.
  • After hybridization, the glass slide is sent for image analysis.

3. Data Analysis (Bioinformatics)

  • Data from the experiment is stored in .CEL files (binary format).
  • Software: R and RStudio are used for data analysis.
  • Affymetrix data is also provided in .CEL format.

License

This repository is licensed under the MIT License.