Understanding Transcriptomics and Gene Expression

• Transcriptomics involves studying all mRNA in cells to analyze gene expression.

Gene Expression Overview

• Gene expression involves multiple steps.
• DNA (genome) in the nucleus is transcribed into pre-mRNA, then mRNA.
• mRNA (transcriptome) moves to the cytoplasm and is translated into proteins (proteome).
• Proteins then influence metabolites (metabolome).
• Regulation also plays a role in gene expression to control mRNA.
• Structural genomics determine the genome map, sequence, and annotations.
• Functional genomics determines protein sequencing, translation, detection, and enzyme activities.

Transcriptome

• Transcriptome is the complete set of mRNAs in a cell, tissue, or organ.
• mRNA levels are influenced by transcription and mRNA stability.

Transcriptomics

• Transcriptomics analyzes gene populations and differences in gene expression under different conditions.

Gene Expression Profiling

• Gene Expression Profiling evaluates the expression of all genes in a system within a specific experimental context.
• The "system" can refer to cells or tissues.
• Gene expression profiling identifies changes in gene expression from conditions to infer their impact on the system.
• Gene expression profiling is a diagnostic tool to understand how changes to a system may impact its function.
• Gene expression profiling compares two conditions which include control vs experimental.
• Such as: wild-type vs mutant or no drug vs drug.

Techniques

• Select Genes/transcripts include transcriptional reporter systems and reverse transcription (RT)-PCR.
• Whole genome/transcriptome methods include microarray and RNA sequencing (RNA-seq).

Analysis of Gene Transcription

• Analysis of gene transcription at the mRNA level uses methods like macroarrays, microarrays and Northern blotting.
• Can be based on hybridization using mRNA (transcriptome) to create proteins (proteome).
• Real-time PCR, qRT-PCR, and semi-quantitative RT-PCR use methods based on PCR.
• Transcriptional fusion of a gene promoter with a reporter gene is also used.

Transcriptional Reporter Systems

• Transcriptional Reporter Systems include a promoter region that drives a reporter gene.
• The reporter gene is used to measure levels of transcription or translation through a transcription/translation process.
• Luciferase, upon interaction, emits light of a certain wavelength.
• Reporter systems are genetic engineering techniques used to study gene regulation.
• Reporter systems are not high-throughput and do not require RNA purification.
• Reporter genes include luciferase (luminescence), GFP (fluorescence), and β-galactosidase (colorimetric or luminescence).

RT-PCR

• RT-PCR is a technique that converts RNA into cDNA and then amplifies the cDNA by PCR.
• Single-stranded cDNA is used as a template.
• Gene-specific primers (GSP) are used.
• RT-PCR can be quantitative and monitored in real-time via qRT-PCR.
• qRT-PCR uses fluorescent dye (e.g., SYBR-Green) or labeled probes (e.g., TaqMan®) to track amplification.

RNA Isolation

• RNA isolation method depends on the source of RNA.
• RNA isolation involves mechanical or chemical disruption of tissues or cells like homogenization or lysis.
• RNA stabilizing agents must be added.
• These agents contain phenol, guanidine isothiocyanate, detergents, and chelating agents to inactivate endogenous RNases and denature proteins.
• Some methods use phenol:chloroform extraction.
• TRIzol® reagent is an example of phenol:chloroform extraction.
• Column-based or resin-based purification kit systems are a common RNA isolation method.
• It is crucial to maintain an RNase-free environment during RNA isolation.

cDNA Generation

• cDNA is complementary DNA.
• Reverse transcription is the conversion of an RNA template to cDNA.
• Special polymerase called reverse transcriptase (RT) is used.
• This is derived from viruses.
• Reaction requires RNA template, RT enzyme, buffers, dNTPs, and primers.
• In the process:
• Oligo dT primers bind to the poly-A tail of mRNA
• Random oligomers are short randomized hexamers that bind to random sequences on the RNA
• Gene-specific primers (GSP) bind to a specific target gene and are used for analyzing a specific transcript

Microarray Technology

• Microarray Technology detects the presence and abundance of nucleic acids in a biological sample.
• Microarray Technology uses a solid surface, like a glass slide, onto which DNA molecules have been chemically bonded.
• Microarray Technology can use spotted DNA arrays and high-density oligonucleotide arrays like GeneChip® by Affymetrix.
• Arrays are designed to be specific for the organism or cell line.
• Each "spot" has multiple DNA probe copies that are complementary to a specific gene or transcript.
• Used for gene expression profiling, SNP identification, and genome re-sequencing.

DNA Arrays and Chips

• Flourescent signal is used to determine intensity and position on DNA array.
• Hybridization is performed.
• An array or chip with an immobilized probe is used.
• Identity and amount of DNA sample are measured.

Terminology

• Macroarrays involved printing of oligo-nucleotids or PCR fragments with a membrane support and max density of 64 probes per cm^2
• Microarrays involved printing of oligo-nucleotids or PCR fragments with a glass support and up to 10,000 probes per cm^2.

Spotted Array

• cDNA Preparation and Hybridization- cDNA samples are labelled with Cy3 (green) and Cy5 (red).
• Labelled DNAs are heat-denatured, applied to the array, and allowed to hybridize.
• Array is washed to remove unhybridized probes.
• A laser excites fluorophores, and fluorescent emission wavelengths are measured.

GeneChip®

• Preparing a GeneChip® from a sample: ss cDNA → ds cDNA → aRNA → labelled with biotin-labeled NTPs, and purified then fragmented.
• Chip is then washed, stained using streptavidin-conjugated fluorophore, and visualized by excitation.

Considerations for Microarrays

• High concentrations of RNA template are needed.
• Experiments should be done with at least two replicates.
• Measures must be taken to limit RNA and DNA contamination.
• Experiments should include internal positive/negative controls with probes from different species on the array.