Transcriptonomics Flashcards
What is the transcriptome?
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”)
What are transcriptomics?
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)
Genomics and transcriptomics address the same class of bio molecules…
- the nucleic acids
- Simple (linear), highly informative structure
- Similar chemical properties that can be effectively used for automation
Explain the value of transcriptonomics
- 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
What are the methods to detect single gene transcriptional genes?
- Hybridization based
- PCR based
- Reporter gene-based
State what is hybridization-based detection of single gene transcriptional changes
Hybridization based
Northern blotting
- transcript-specific radioactive probes are used to identify a target mRNA species within an immobilized RNA sample
How are PCR based techniques able to detect single gene transcript ?
- 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
How can reporter gene-based techniques be used to detect single gene transcripts be used ?
- 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
What are the PCR techniques used to detect multiple gene transcriptional changes?
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
What are the hybridization-based techniques used to detect multiple gene transcriptional changes?
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
What methods are used to detect multiple gene transcriptional changes?
PCR-based
Hybridization based: microarrays, macroarrays
Sequencing-based: SAGE (Serial Analysis of Gene Expression, MPSS( Massively Parallel Signature Sequencing)
How does Sequencing based techniques detect multiple gene transcriptional changes?
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
Explain the functioning of Sequencing-based methods: SAGE (Serial Analysis of Gene Expression, MPSS( Massively Parallel Signature Sequencing
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
Explain the functioning of Massively Parallel Signature Sequencing (MPSS)
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
MPSS is possible …
Without organism sequence information
-MPSS can accurately quantify transcripts as low as 5 transcripts per million (tpm) to above 50,000 tpm
What are expression arrays?
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
What are the types of expression arrays?
- SNP chips
- point mutations - Microarrays
- gene expression - Tiling Arrays
- expression across a genome or chromosome
What are microarrays?
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
What are the basics of microarray?
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
Give the method of interpretations of microarray
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
What is the function of the Affymetrix Gene chip?
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
What are the steps to perform a two-channel microarray?
- Isolation of two tissues
- Isolation of RNA
- Production of labeled cDNA
- Hybridization of labeled cDNA
- Visualization of hybridization
What is a two-channel microarray?
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
Describe imaging of expression profile
-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
