BCH 4101 Lecture 11: Transcriptome Analysis
28 Apr 2018
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November 20, 2017
Transcriptome Analysis
Transcriptome
Complete set of RNA transcripts (mRNA, ncRNA) that are produced by the genome, under specific circumstances or
in a specific cell
-Studied using high-throughput methods
-Definition has changed over time
mRNA fraction: small, but complex (<5% of all RNAs in a cell)
10 000 - 15 000 genes expressed in a single tissue (cerebellum and testes are most complex)
-These thousands of genes are able to express multiple proteins
Alternative splicing, multiple start/end points —> individual gene can give rise to several mRNAs
Why do we want to study the transcriptome?
Important for understanding human health and disease
-Ex. Can help figure out different proteins that are expressed in cancer cells for drug development purposes
Understand human development
What protein and RNA are needed to make and keep functioning of different specialized cells
Understand the activation pathway of function for a given stimulus
Understand the relationship between genes based on their shared or unshared pattern for expression
Understand the unique expression profile of a gene (gene signature)
Interpret the functional elements of the genome
Transcriptomics
Key aims of transcriptomics:
-Catalogue all species of transcript (mRNA, ncRNA, etc.)
-Determine the transcription structure of genes (start site, 5’ and 3’ ends, splice patterns, etc.)
-Quantify the changing expression levels of each transcript
Change of RNA levels can show effect of treatment
Real-Time PCR
qRT-PCR:
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November 20, 2017
-Reverse transcription of mRNA template into cDNA
•Isolate mRNA fraction from total RNA and convert it into cDNA
-Can convert in many different ways (ex. Oligo DT primer, can bind to poly-A tail in mRNA)
•Need gene-specific pair of primers that will be used to synthesize complementary DNA strands
-Primers usually span an exon-intron boundary
-If primers don’t span boundary, they can bind to genomic DNA that contains an intron
-Amplification of cDNA
•Amplification from cDNA increases specificity and decreases amplification from contaminants
-Measurement of fluorescent signal in real-time (proportional to the amount of DNA present)
•Can measure how much DNA is being produced and is in your tube after each round
•Amplification is exponential
•Fewer copies will give a weaker signal, and requires more cycles before the gene or interest can be detected
•Can be used to estimate mRNA copy number of individual mRNAs by comparing the results to a standard curve
for that particular mRNA
DNA-binding dyes (SYBR Green): binds non-specifically to dsDNA
-Fluorescent signal increases 1000x when bound to dsDNA (no fluorescence when just in soluution)
-Advantage: simple primer design
-Disadvantage:
•Lack of specificity: binds to all dsDNA
•Can’t multiplex: can’t look at multiple genes because you wouldn’t know where each gene makes up the part of
fluorescence
•Primer dimers can occur: primers can bind to each other and the dimer can be bound by dye, which will give a
false fluorescence reading
Probe-based detection
-Hydrolysis (Taqman) probes: sequence-specific probe
•Design PCR primers and design a probe that is specific to the gene of interest
•Fluorescently quenched so that they don’t emit a signal when not found
-Reporter nucleotide is bound to the quencher nucleotide
-Taq polymerase can cleave between these two nucleotides
-During extension, reporter is freed from quencher, emits signal, and the signal can be measured
•Exploit 5’—>3’ exonuclease activity of polymerase cleaves the probe
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Document Summary
Complete set of rna transcripts (mrna, ncrna) that are produced by the genome, under speci c circumstances or in a speci c cell. De nition has changed over time mrna fraction: small, but complex (<5% of all rnas in a cell) 10 000 - 15 000 genes expressed in a single tissue (cerebellum and testes are most complex) These thousands of genes are able to express multiple proteins. Alternative splicing, multiple start/end points > individual gene can give rise to several mrnas. Can help gure out different proteins that are expressed in cancer cells for drug development purposes. What protein and rna are needed to make and keep functioning of different specialized cells. Understand the activation pathway of function for a given stimulus. Understand the relationship between genes based on their shared or unshared pattern for expression. Understand the unique expression pro le of a gene (gene signature) Catalogue all species of transcript (mrna, ncrna, etc. )
