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Molecular Biology > Transcriptonomics > Flashcards

Transcriptonomics Flashcards

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1
Q

What is the transcriptome?

A

mRNA that is present in a cell at a particular time

Percentage of the genetic code that is transcribed into RNA molecules (depends on development, environment, time of the day, tissue)

Collection of all gene transcripts present in a given cell/tissue at a given time (“snapshot”)

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2
Q

What are transcriptomics?

A

Global analysis of gene expression= genome-wide expression profiling

mRNA Levels compared in Different Contexts

  • different tissues, same organism (brain v. Liver)
  • Same tissue, same organism (tumor vs non-tumor)
  • Same tissue, different organisms (wild type v. Mutant)
  • Time course experiments (development)
  • Other special designs (e.g. to detect spatial patterns)
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3
Q

Genomics and transcriptomics address the same class of bio molecules…

A
  • the nucleic acids
  • Simple (linear), highly informative structure
  • Similar chemical properties that can be effectively used for automation
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4
Q

Explain the value of transcriptonomics

A
  • Used to identify the mRNAs and determine relative abundance
  • Gives an understanding of genes and pathways involved in biological processes (“guilt by association”, that is, genes with similar expression may be functionality related and under the same genetic control mechanism )
  • Helps elucidate the function of unknown genes based on their spatial and temporal expression
  • Identifies marker genes for diagnosis of diseases
  • Gene expression allows indirect inferences about genetic differences
  • May be proxy for changes in the proteome and metabolome
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5
Q

What are the methods to detect single gene transcriptional genes?

A
  • Hybridization based
  • PCR based
  • Reporter gene-based
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6
Q

State what is hybridization-based detection of single gene transcriptional changes

A

Hybridization based
Northern blotting

  • transcript-specific radioactive probes are used to identify a target mRNA species within an immobilized RNA sample
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7
Q

How are PCR based techniques able to detect single gene transcript ?

A
  • Based on the ability of a PCR to exponentially amplify initial differences in transcript number
  • amplified products are visualized either in real time during (qPCR, A) or after (RT-PCR, B) the reaction
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8
Q

How can reporter gene-based techniques be used to detect single gene transcripts be used ?

A
  • fusions of a promoter of a gene of interest (GUS), green fluorescent protein, luciferase
  • reporter activity is measured histochemical
  • allows for detailed spatial and kinetic analysis
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9
Q

What are the PCR techniques used to detect multiple gene transcriptional changes?

A

Differential display RT-PCR, cDNA-AFLP (Amplified Fragment Length Polymorphism )

  • reverse transcription of mRNA into cDNA
  • divide cDNA pool into subsets by selective PCR amplification
  • separation of sub pools on high resolution gels
  • quantification of band intensity
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10
Q

What are the hybridization-based techniques used to detect multiple gene transcriptional changes?

A

Macroarrays, microarrays

  • a small membrane (microarray) or glass slide(microarray) containing samples of many genes arranged in a regular pattern
  • mRNA samples of interest are fluorescent labelled and either singly or competitively hybridized to a slide
  • in a single experiment, the expression levels of thousands of genes can be determined by measuring the amount of mRNA bound to each gene on the array
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11
Q

What methods are used to detect multiple gene transcriptional changes?

A

PCR-based

Hybridization based: microarrays, macroarrays

Sequencing-based: SAGE (Serial Analysis of Gene Expression, MPSS( Massively Parallel Signature Sequencing)

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12
Q

How does Sequencing based techniques detect multiple gene transcriptional changes?

A

Sequencing-based: SAGE (Serial Analysis of Gene Expression, MPSS( Massively Parallel Signature Sequencing

  • short sequence signatures produced from a defined position within an mRNA
  • the relative abundance of these signatures (tags) in a given library represents a quantitative estimate of expression
  • no sequence knowledge required! Universal platform to study any transcript
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13
Q

Explain the functioning of Sequencing-based methods: SAGE (Serial Analysis of Gene Expression, MPSS( Massively Parallel Signature Sequencing

A

SAGE generates sequences from cDNA fragments for the discovery of new genes and the quantification of their expression levels in a certain tissue

  • Isolate mRNA from a sample (e.g. a tumor)
  • Make cDNA from the isolated mRNA
  • Extract a small chunk of sequence from a defined position of each cDNA molecule
  • Link these small pieces of sequences together to form a long chain (or concatemer)
  • Concatemerized tags are sequenced using a traditional automated DNA sequencing method
  • Process this data with a computer to count the small sequence tags
  • tags are ~9 to 14 bp in length
  • Library may contain ~50,000 tags
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14
Q

Explain the functioning of Massively Parallel Signature Sequencing (MPSS)

A

cDNA fragments are cloned onto micro-beads using the Lynx Megaclone technology

  • Starting with one million mRNA molecules from a particular cell or tissue sample, Megaclone will produce one million beads, each containing 100,000 cloned copies of cDNA from each mRNA molecule
  • All molecules are covalently attached to the micro-beads at their poly(A) ends, so the Dpnll end is available for the sequencing reactions
  • Uses a novel sequencing method whereby thousands of sequences are obtained simultaneously by sequencing off of beads
  • Captures and identifies transcript sequences of expressed genes by counting the number of individual mRNA molecules representing each gene
  • Individual mRNAs are identified through generated 17- to 20-bade signature sequence
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15
Q

MPSS is possible …

A

Without organism sequence information

-MPSS can accurately quantify transcripts as low as 5 transcripts per million (tpm) to above 50,000 tpm

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16
Q

What are expression arrays?

A

A cDNA array containing features representing a set of known expressed sequences

-Used to identify a set of genes or all genes that are (expressed)transcribed in a cell or tissue

17
Q

What are the types of expression arrays?

A
  1. SNP chips
    - point mutations
  2. Microarrays
    - gene expression
  3. Tiling Arrays
    - expression across a genome or chromosome
18
Q

What are microarrays?

A

A cDNA microarray is based on the ability of a given mRNA molecule to bind specifically to(hybridize to) its original DNA coding sequence in the form of a cDNA template spotted on an array

19
Q

What are the basics of microarray?

A

Technologies:

  • robotic spotting (ESTs, oligonucleotides)
  • in-situ synthesis (oligonucleotides), Affymetrix, NimbleGen, Agilent

Hybridization:

  • competitive in two color arrays
  • single in one color arrays

Cross-Species Hybridization:
-Gene probes and samples originate from different species

20
Q

Give the method of interpretations of microarray

A

Cy signal~ amount of mRNA in healthy & diseased tissue

Green spots= cDNA from healthy tissue hybridized to the target DNA

Red spots= cDNA from diseased tissue hybridized to the target DNA

Yellow spots= control and sample cDNA hybridized equally to the target DNA

Blue spots= neither control nor sample cDNA hybridized to the target DNA

21
Q

What is the function of the Affymetrix Gene chip?

A

Probes are synthesized on a chip

Probes are oligonucleotides of a specified length
-Generally 25 times

At each x,y location a particular oligonucleotide is synthesized in 1000s of copies at that location

One chip per sample

  • One for control
  • One for each experiment

When the tagged (red ball) RNA is added to the array, the RNAs “look” for their complimentary match

22
Q

What are the steps to perform a two-channel microarray?

A
  • Isolation of two tissues
  • Isolation of RNA
  • Production of labeled cDNA
  • Hybridization of labeled cDNA
  • Visualization of hybridization
23
Q

What is a two-channel microarray?

A

Similar microarray technology is being used to identify potential gene targets for therapies to treat human disease

For example, which genes are expressed in cancerous tumors compared to non-cancerous tumors

24
Q

Describe imaging of expression profile

A

-Two-channel microarray in which cDNA samples from two different tissues are competing for binding to the same probe set

Rinse off excess cDNA, put the microarray in a scanner to measure fluorescence of each spot. Fluorescence intensely indicates the amount of mRNA expressed in the tissue sample

25
Q

What are the standards for Microarray Experiment?

A

MIAME is the internationally adopted standard for the Minimal Information About a Microarray Experiment

The result of an MGED: Microarray Gene Expression Data Society (now FGED: functional Genomic Data Society) driven effort to codify the description of any microarray experiment

MAIME aims to define the core that is common to most microarray experiments

Ultimately, it tries to specify the collection of information that would be needed to allow somebody to interpret the results of an experiment unambiguously and to completely reproduce the experiment that was performed elsewhere

26
Q

What are the applications of microarray?

A

Medicine-

  • disease-associated expression patterns (diagnosis)
  • Cell-cycle monitoring(cancer research)
  • Treatment-induced expression pattern (drug development and response)

Biology

  • Development and Morphology(juveniles vs adults, male vs female, tissue 1 vs tissue 2)
  • Interactions between organisms (antagonistic, mutualistic, competitive) and organisms and their environments (temperature, radiation, drought, nutrient levels, toxins and heavy metals)
  • Evolution (within and between species variation, hybrids vs parents )
  • Functional analysis (wild type vs mutant)
  • Time series analysis
27
Q

What is a Tiling Array?

A

-SNP chip and Microarray expression profiling applications use probes biased toward known and predicted gene structures

Tiling arrays are made up of high-density probes; use array based hybridization to scan

  • particular region(s) of the genome
  • the entire human chromosome
  • the entire genome of an organism
  • Genome array consists of overlapping or non overlapping probes
28
Q

What is the methodology/value of Tiling Array?

A

-Characterize regions of genome which are sequenced but with local functions that are largely unknown

-Used in identifying novel transcripts, exons which may not have been previously predicted
     mRNA
    -Alternative splicing
    - Not Polyadenylated 
     miRNA

Tiling arrays- 1 million spots representing DNA sequences spread across entire chromosomes

By labeling cDNAs derived from cells it is possible to identify regions of chromosomes that are transcriptionally active

-Used to identify areas of the genome not previously thought to contain genes

29
Q

What Gene expression detection assays used for cancer ?

A
  • TMA
  • FISH
  • NanoString
  • qRT-PCR
  • Microarray
  • RNA-seq
30
Q

How are TMAs used for cancer?

A

Hundreds samples, FISH

Tissue microarrays (TMAs)-study circular punches from tissue sample blocks with labelled probes or antibodies to determine the gene expression

31
Q

How can FISH be used for cancer?

A

Single sample, localization and hybridization intensity

32
Q

How can NanoString be used for cancer?

A

Probes hybridization intensity 50-500 genes

NanoString nCounter uses probe pairs to anneal to a region of RNA and detect genes

33
Q

How are qRT-PCR used for cancer?

A

Reaction cycle number to reach the optimal slope, 1-50 genes

Amplifies a gene of interest and uses fluorescent probe or dyes to depict the gene expression

34
Q

How are Microarray used for cancer?

A

Hybridization intensity 1000-25,000 genes

DNA microarrays allow cDNA targets to hybridize to probes on a solid slide and can use fluorescence to detect gene

35
Q

How are RNA-seq been used for cancer?

A

Transcripts numbers

Whole transcriptome

Illumina MiSeq RNA-Seq bridge amplifies nucleic acid samples to create clusters, whichever then interpreted by the MiSeq system

Molecular Biology (24 decks)

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