Chapter 10: Analysis of Gene Expression: Transcription

Targeted Approach

Transcript is known, and we want to know abundance/localization

• Northern Blot
• In-situ Hybridization
• Semi-quantitative PCR

Untargeted Approach

looking for differences amongst transcripts profiling/monitoring

• EST Abundance
• Transcriptome Sequencing
• Microarrays
• Subtractive Hybridization

Genes respond to more than 1 factor and can be expressed constitutively, spatially, temporally

Constitutive: expressed at all times in every cell but may change level of expression (actin)

Temporal: expressed at different times in it’s development, controlled by inducible promoter (all or some cells)

Spatial: some genes expressed in specific tissues & not others, inducible promoter

Most techniques measure steady-state level of transcript abundance: a reflection of mRNA synthesis rate and rate at which it is degraded (turnover)

Detection of Transcripts: How rare is transcripts, what tools do you have etc.

Northern Blot

Purpose

• Detect presence of transcripts similar to probe sequence in a population of mRNA
• Detect changes in transcript size
• Detect relative abundance of transcript in different samples
• Must ensure that same amount is loaded, and RNA is intact
• Denaturing agarose gel (formaldehyde) to prevent RNA from folding back on itself, allowing migration by length
• Size markers of known RNA in one lane, mRNA is only 1% of total RNA, faint smear
• Darker bands are ribosomal RNA much more abundant (check to see if RNA’s intact)
• Intensity of signal relates to abundance of target transcript

Controls

test if RNA equally loaded and intact, test for equal loading on the gel (bottom) and the membrane is on top, if blot blank then problem with probe, test total RNA equal by demonstrating consistently expressed common mRNA like acting, use spectroscopy to measure amount of RNA

Can Monitor multiple samples simultaneously, detect size of transcript, tell is probe is specific, miRNA detection and cheap but only provides elative abundance, not very sensitive, must make a probe, several days

In Situ Hybridization

Used for spatial expression analysis to determine which cells express transcript, do a northern blot first to learn probe specificity, antisense probe used to bind the mRNA, use sense strand as control (should not bind), it will bind anything that sticks to RNA

• Fix tissue of interest (to kill RNase) with chemicals and embed in plastic/wax
• Cut tissue into thin sections with microtome, hybridize sections with riboprobe labelled DIG, bound probe is detected

Controls

Hybridize with sense to see regions binding non-specifically, no probe watch for colour, no probe but add antibody and see antibody specificity

Can localize transcripts within few cells but must verify hybridization conditions are gene specific by northern blotting first, not a quantitative technique, make probe specific to UTR of gene to ensure gene specific

RT-PCR

• A sensitive assay for estimating transcript abundance and quantification of similar sequences (faster and more specific than Northern Blot)
• When done in a semi quantitative mode, exponential phase of PCR reaction, intensity of RT-PCR product indicates abundance of corresponding transcript in population used for reaction
• Isolate mRNA from cells of interest and reverse transcribe using oligo-dT primers
• Use mixture of cDNA as template for PCR & quantify product during exponential phase
• Need antisense and sense primers for PCR, for RT PCR use one primer with UTR and one with gene family region

Controls

Use template that binds primers for sure, leave out template and test for contamination, use a mutant without gene to test primer specificity, leave out RT to test genomic DNA contamination, design primers that produce a product that span an intron, so if gDNA present larger PCR product

Gene specific detection, no hybridization necessary, can sequence the product, very sensitive requires less RNA but difficult to be sure of quantification without controls, real time or semi quant are used for exact quantification,

Regular PCR isn’t quantitative because after certain # of cycles product stops doubling when primers and dNTPs are limiting and saturated, exponential phase occurs in the PCR cycled when reaction is in excess.

Semi Quant: Transcript abundance relative to other samples, look at difference in 2 fold increase in both

Quantitative RT-PCR

Specialized machine, bottom of each well is transparent and when the plate is in the machine the amount of fluorescence produced by DNA synthesis is monitored as the reaction occurs, making it possible to obtain an estimate of transcript abundance, can use a dye that binds double stranded DNA, or fluorescent molecule on primer

Rules

1. Harvest material from 3 biological replicate, use isolation method for high-quality total RNA
2. Monitor quality and quantify, treat with DNase1 to remove gDNA and do trial PCR
3. Use high quality RT and good ply-dT primer, do trial RT-PCR with primers for known genes
4. Design gene-specific primers and reduce pipette and technical errors
5. Test 4 different reference genes that are stably expressed in all the samples and preform qPCR on reference genes and test samples in parallel
6. Determine which reference gene is the best and calculate relative transcript abundance

Comparing Transcriptomes

Transcriptome: Complete mRNA complement of a cell, goal is to determine which transcripts are expressed at the same time or change in a specific mutant in response to a treatment, often an early step to identify important genes

Expressed Sequence Tags: Only partial sequence of cDNA but includes a UTR so identifies specific gene

• cDNA libraries generated using mRNA isolated from cells or organism, to identify which cDNA present many random clones from each library are partially sequenced for the UTR and part of coding region
• Transcripts are identified using BLAST, done on a large scale with #s of each transcript, transcript profile
• Number of ESTs for a gene = number of transcripts, provide an estimate of range of transcripts in cell type
• At the end, sequence data and store in database, can recover cDNA on sequence info by PCR, compare transcriptome sequence to genome sequence and determine intron/exon

Transcriptome Sequencing

• Massively parallel sequencing, many DNA fragments are sequenced at a time creating a cDNA library but not cloning into a vector, completely sequence components with parallel sequencing methods
• Deep-sequencing: detecting rate transcripts and splice variants and transcriptome differences amongst cells as well as miRNA and antisense strands
• Wide dynamic range can measure lower and higher abundance, no saturation in parallel sequencing but must control for all other conditions and many replicates for statistical significance
• Verify quality of sequencing data, map to genome or transcriptome, normalize data, detect differential expression, functional analysis and validate results by semi-quantitative PCR

Microarrays

• Oligonucleotides correspond to sequence of genes fixed on a chip
• Tilling Arrays: Short overlapping oligoNT that extend across chromosome & monitor genome
• cDNA arrays: cDNAs purified and spotted by robot onto the glass slide
• Used to compare transcript abundance of many genes simultaneously
• Signal from bound probes is visualized by fluorescence, add differently labeled cDNA from 2 sample populations to observe what is in common, all ORF can be monitored in 1 experiment
• Can include every gene or focus on specific group, use a ribonucleotide with flu tag to emit

Control

Sequences that should not hybridize/genes not in population tested , switch colours as some label better, use sequences that should hybridize and validate with nblot or rtpcr / qrtpcr

See many expression changes & glimpse of transcriptional activity, tilling arrays reveal anti sense transcripts, data set is publicly available , can see what genes change in same patter as gene of interest but not gene specific (cDNA share ORF amongst family, tilling arrays are gene specific), microRNAs not detects, several replicates required, generate large amounts of data, transcriptional changes do not reflect protein abundance or metabolic activity

Subtractive Hybridization

Mix 2 populations of cDNA, subtract transcripts in common, not profiling method, differential screening

Purpose

Detect and recover transcripts that differ between two closely matched mRNA populations

• Solution hybridization, two mRNA populations converted to cDNA and one population has a primer with an affinity tag (driver), driver is in excess to tester often done in both directions
• cDNA is mixed, denatured and allow to hybridize, mixture is passed over affinity column and only sequences unique to untagged population will be cloned because others bind to column
• These sequences are recovered cloned and sequenced by sanger

Distinguishing between Transcript Initiation and mRNA stability

If differences in transcript abundance are observed, they may be due to differences in initiation or stability

Monitoring Transcription Initiation

If no differences then may be due to turnover

Nuclear Run on Assays

• Measure transcript abundance in nucleus, only promoter activity because degradation in cytosol
• Isolate nucleic acid from cells expressing transcript (difficult), chemically inhibit new transcription initiation but let started ones complete
• Look at nascent transcripts – newly started and incubate the nuclei with 32P UTP to label these transcripts, this pool of mRNA is darioactive and used as a probe for dot blot of specific genes
• Hybridize it to an excess blot with genes of interest

Controls

Include spots for transcripts known to be up or down regulated

Readily compared to northern blot and qrtpcr but difficult to isolate nuclei and danger in radioactive

Transcriptional Fusions

• Reporter gene encodes a product that can easily be assayed, replace coding region with reporter coding region and measure promoter activity
• Not a mutant, just has an extra promoter regulating color product which is harmless

Reporter Genes

lacZ

• Signal: Blue
• Advantage: Microbial hosts, stable cheap
• Disadvantage: Very Stable – not good for dynamic changes

Luciferase

• Signal: Light
• Advantage: Short half-life, sensitive to change
• Disadvantage: Expensive, special camera

GUS

• Signal: Blue
• Advantage: Cheap and Easy
• Disadvantage: Protein too stable, last longer than transcript

GFP YFP RFP

• Signal: Fluoresce
• Advantage: No extra substrate, organism alive
• Disadvantage: Specialized microscope

Different Experiments: Test when a promoter active, fuse promoter active in presence of certain metabolite, fuse promoter active in condition & mutagenize the organism look for no signal (mutant in protein controlling promoter)

Limitations: Transformable organism, some signals too stable, reporter may diffuse to other cells, most kill organism, so difficult to do developmental studies

Investigating Cis Elements in Promoters

• Consider if promoter fragment chosen contains all relevant cis elements, is complete promoter used in reporter gene then reporter gene will be expressed exactly as gene of interest, first detection analysis
• Idea of where promoter might be: distance between adjacent genes, bioinformatics
• Search for cis elements using computer programs identifying palindromic repeat sequences , test using deletion analysis
• Control: Ensure expression or promoter matches endogenous gene

In vivo assay allows detection of transcription factors but takes a lot of work and must make many transformants, sometimes the reporter gene too stable, sometimes regulatory sequences away from coding region, reporter gene may diffuse