Transcriptome analysis; Proteomics

Question 1

Transcriptome analysis attempts to

Answer 1

catalog and quantify the total RNA content of a cell, tissue, or organism.

Question 2

Transcriptome analysis reveals gene-expression profiles that, for the same genome, may

Answer 2

vary from cell to cell or from tissue type to tissue type

Question 3

Transcriptome analysis provides insights into:

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■ normal patterns of gene expression that are important for understanding how a cell or tissue type differentiates during development
■ how gene expression dictates and controls the physiology of differentiated cells
■ mechanisms of disease development that result from or cause gene-expression changes in cells.

Question 4

For example, examining gene-expression profiles in a cancerous tumor can help diagnose tumor type, determine the likelihood of tumor metastasis (spreading), and develop the most effective treatment strategy.

Question 5

Transcriptome analysis also called

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transcriptomics or global analysis of gene expression

Question 6

Transcriptome analysis studies the expression of genes in a genome

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both qualitatively and quantitatively.

Question 7

Qualitatively

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• by identifying which genes are expressed or not expressed.

Question 8

Quantitatively

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• by measuring varying levels of expression for different genes.

Question 9

For nearly two decades DNA microarray analysis has been widely used because it

Answer 9

enables researchers to analyse all of a sample’s expressed genes simultaneously.

Question 10

A single microarray can have over 20,000 different spots of DNA, each containing

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a unique sequence that serves as a probe* for a different gene.

Question 11

Probe

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need to have sequence information about the genes of interest

Question 12

PCR-based methods - such as reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qPCR) are useful because

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of their ability to detect genes expressed at low levels.

Question 13

Now, RNA-Seq has superseded arrays – totally dominate expression analysis.

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Qualitative and quantitative for totally unknown samples.

Question 14

Microarray Analysis

Question 15

DNA Microarray Analysis – How does one interpret an experiment?

In this experiment a microarray was used to analyse gene expression patterns as a plant was infected with a pathogen – over a period of 290 min (4.8 hours). Assume it was an Arabidopsis plant and we used the Affymetrix gene chip (picture to the right). Looking more closely we see on the chip (picture on the left), specific clusters of genes being up- or down-regulated as the infection progresses. This gives us valuable information as to how Arabidopsis responds to a pathogen infection.

Question 16

Microarrays are limited

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As they can only study the expression of the genes with probes on the chip.

Question 17

Direct RNA sequencing (RNA-seq) - called whole-transcriptome shotgun sequencing - allows for

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quantitative analysis of all RNAs expressed in a particular tissue and, also provides actual sequence data.

Question 18

Proteomics

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the identification, characterisation, and quantitative analysis of proteomes

Question 19

The Proteome is defined as

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the complete set of proteins encoded by a given genome

Question 20

Studying the proteome can yield valuable information about:

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A protein’s structure and function
Posttranslational modifications
Protein-protein, protein-nucleic acid, and protein-metabolite interactions
Cellular localisation of proteins
Protein stability and aspects of translational and post-translational levels of gene-expression regulation
Relationships (shared domains, evolutionary history) to other proteins

Question 21

Proteomes are larger than genomes

Answer 21

[Sequencing of mRNAs from human tissues found that over 95 % of protein-coding genes with more than one exon are alternatively spliced.]
Different transcriptional start sites
Alternative mRNA splicing
RNA editing
Post-translational modifications (> 100 mechanisms are known)
Human Proteome Map (HPM) – to cataloque the human proteome (2014)

Question 22

About __________ of the Drosophila proteome has been well catalogued using proteomics.

Answer 22

two-thirds

Question 23

Proteomics is also of clinical value because it

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allows comparison of proteins in normal and diseased tissues, which can lead to the identification of proteins (or smallRNAs) as bio-markers for disease conditions.

Question 24

Genes can have multiple transcription start sites that produce several different types of RNA transcripts.

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Alternative splicing and editing of pre-mRNA molecules can generate dozens of different proteins from a single gene.

Question 25

determined in large part by its gene-expression patterns

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The specific protein content (or profile) of a cell - its transcriptome

Question 26

However, a number of other factors affect the proteome profile of a cell.

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To begin with, many proteins undergo co-translational or posttranslational modifications, such as cleavage of a signal sequence that targets a protein for an organelle pathway, a propeptide, or initiator methionine residues; linkage to carbohydrates and lipids; or the addition of chemical groups through methylation, acetylation, and phosphorylation; and other modifications. Over a hundred different mechanisms of posttranslational modification are known.

Question 27

Early Proteomics - Two-dimensional gel electrophoresis (2DGE)steps

Answer 27

1. Proteins are isolated from cells or tissues and first loaded onto a polyacrylamide tube gel and separated by isoelectric focusing, which causes proteins to migrate based on their electrical charge in a pH gradient.
2. During isoelectric focusing, proteins migrate until they reach the location in the gel where their net charge is zero relative to the pH of the gel.
3. In a second migration, perpendicular to the first, the proteins are separated by their molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
4. Proteins are identified by cutting spots out of a gel and sequencing short stretches of the amino acids that the spots contain.
5. BLAST can then be used to search protein databases containing amino acid sequences of known proteins.
6. However, because of alternative splicing or posttranslational modifications, peptide sequences may not always match easily and, the protein may have to be confirmed by another approach.

Question 28

Two-dimensional gel electrophoresis (2DGE)

Question 29

Mass spectrometry techniques analyze

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ionized samples in gaseous form and measure the mass-to-charge (m/z) ratio of the different ions in a sample

Question 30

Proteins analyzed by mass spectrometry generate m/z spectra that can be correlated with an

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m/z database containing known protein sequences to discover the protein’s identity.

Question 31

Some of the applications of Mass spectrophotometry (MS) are to:

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Identify an unknown protein or proteins in a complex mix of proteins
Sequence peptides
Identify posttranslational modifications of proteins
Characterize multiprotein complexes.

Question 32

One common MS approach is matrix-assisted laser desorption ionization (MALDI).
This approach is ideally suited for

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identifying proteins and is widely used for proteomic analysis of tissue samples.

Question 33

MS MALDI

Answer 33

MALDI employs an ultraviolet laser to heat, vaporize, and ionize peptide fragments. Released ions are then analyzed for mass; MALDI displays the m/z ratio of each ionized peptide as a series of peaks representative of the molecular masses of peptides in the mixture and their relative abundance.

Automated, high-throughput instruments can excise all spots froom a 2D gel for MALDI analysis.

MALDI produces a peptide “fingerprint”, characteristic of the protein being analysed.

Can be compared to MALDI-generated m/z spectra of known proteins.

Question 34

MS MALDI

Question 35

MS - Dinosaurs and birds, distant cousins?

Answer 35

In this example, tissue was harvested from the femur bones of a T-Rex fossil. The T. rex proteins extracted from the tissue showed cross-reactivity with antibodies to chicken collagen and could be digested by the protease, collagenase.
These results suggested that the T. rex protein samples contained collagen, a major matrix component of bone, ligaments, tendons, and skin.
To definitively identify the presence of collagen, peptides from the T. rex samples were analyzed by mass spectrometry.
The m/z spectra for one of the T. rex peptides was identified from a database of m/z spectra as corresponding to collagen.