Lecture 30

Question 1

Measuring changes in gene expression:

Answer 1

• Transcriptomics: from DNA or RNA, genome wide
• Proteomics: under specific conditions, as a proxy for transcriptomics
• Metabolomics: a proxy for gene regulation cell wide or genome wide

Question 2

Transcriptomics:

Answer 2

• Can measure transcription initiation using: RNApol ChIP and nuclear run-on
• Can measure RNA abundance using: northern blot, RT-PCR< microarray, RNAseq
• RNA location : transcriptional fushions

Question 3

Measuring transcript levels:

Answer 3

• Steady state, a balance between transcription and degradation rates
• Probes or primers can be designed to detect pre-RNA, mRNA or both
• Generally use probes or primers that detect the subject

Question 4

Northern blot hybridisation:

Answer 4

• Isolate RNA
• Denature it, so it is linear
• Fraction it according to size through a gel matrix
• Transfer to a membrane support
• Hybridise with a gene specific probe
• One gene at a time
• Can look at multiple transcripts or regulated expression or differential expression

Question 5

RT-PCR or qPCR:

Answer 5

• A measure of the kinetics of amplification of transcripts, measuring how quickly you can make more of a particular transcript of interest
• Isolate RNA
• Convert to cDNA
• Two primers are amplified, the rate at which it increases depends on its starting abundance
• Detection via general dsDNA fluorescent dyes or TaqMan probes measured by a machine

Question 6

TaqMan probes:

Answer 6

• A transcript that is homologous to the transcript of interest
• At one end of the probe there is a fluorescent gene that emits at a particular wave length
• On the other end there is another fluorescent protein
• Light that excites the first probe at particular frequencies will excite the other probe

Question 7

What are you looking for with TaqMan probes?

Answer 7

• Look for the transfer of light between two probes. The close the are the more efficient the transfer
• The TaqMan probe sits between the two primers, the RNAP displaces the probe from the DNA so the energy transfer no longer happens, so you can see it before, but not after transcription

Question 8

Genome-wide transcripts:

Answer 8

• Temporal and spatial expression patterns

- Must map transcripts in all cells, tissues, at all times, in all conditions

Question 9

ENCOE project:

Answer 9

• Identify all functional elements in the human genome
• Sequencing all human DNA transcripts, regulatory elements, histones, replication sites and polymorphisms
• mRNA abundance can be measured, methylation states can be compared, etc.
• Published in 2007

Question 10

Transcriptomics using microarray:

Answer 10

• Use probes like oligonucleotides or PCR products on
• RNA from cell or tissue is hybridised to a set probe plate competitively (cell 1 vs cell 2)
• Fluorescence is measured and transcript levels are determined
• This is a relative value

Question 11

Disadvantageous with microarrays:

Answer 11

• Spotting is not completely clear
• Not quantitative, but is competitive, so good for comparing cell types and measuring relative amounts of sequences
• Predetermining what types you can percieve as you decide what probes to use

Question 12

Types of microarrays:

Answer 12

• EST (spotting cDNAs)
• Gene model coding
• Non-coding (sequences between annotated genes)
• Tiling (probes step along the genome at a fixed different, can identify novel genes)
• Strand-specific (to see which strand the mRNA comes off!)

Question 13

Probe or oligonucleotide:

Answer 13

• Gene specific
• Based on gene annotation or EST
• Unlikely to detect unknown transcripts
• Complicated by differential splicing
• Generally used to measure transcript levels and gene expression

Question 14

Use of microarray base expression profiling:

Answer 14

• Labelled cDNA
• Competitive hybridisation, each horizontal line corresponds to a gene transcript
• Clustered according to similarity in pattern of expression

Question 15

Oligonucleotides and tiled arrays:

Answer 15

• Tiled microarrays are oligonucleotides designed in a step wise manner across a genome
• Can be gapped, designed across both strands, or can be overlapping
• Detects known and novel transcript levels and gene structure
• A gapped tile microarray can allow detection of transcripts and new exons, intron peaks and new genes

Question 16

RNAseq:

Answer 16

• High throughput sequencing of all of the transcripts
• Parallel sequencing of millions of cDNAs with short to moderate read lengths
• Align to reference genome or de novo assembly then alignment using computer programs

Question 17

RNAseq method:

Answer 17

• Take RNA
• Convert to cDNA
• Ligate adaptors that can be placed on a glass slide
• DNA attaches to float cells through covalent linkage with the glass slide
• PCR the glass slide, amplifying up the transcript on a particular cluster
• Sequence the cluster using a primer and addition of nucleotides one at a time, slowly building up a sequence for each cluster