Techniques to Study Gene Regulation

How do we know that transcriptional regulation is a commonly used mechanism for differential gene expression?

• differential gene expression is a used mechanism

• see differences in level of RNA transcripts being produced in different tissues

• differential distribution of transcripts within a developing organism

  • transcripts (mRNA) for only a subset of the genes of a genome are found in a specific cell/tissue/organ type

  • some genes are expressed in all cells, but most are not

Nucleic Acid Hybridization

• hybridization: phenomenal in which single-stranded DNA or RNA molecules anneal to complementary DNA or RNA

• a hbridization probe is a fragment of DNA or RNA which is readioactively or non-radioactively labeled

• it can be used t detect the presence of nucleotide sequences (DNA or RNA) complementary to the sequences in the probe

Methods: Transcript Distribution in an Organism

Several methods can be used to determine the temporal and spatial distribution of gene transcripts during development

Methods for detecting Transcript Distribution: RNA Northern blot

• isolate mRNA from cells from different tissues or developmental times

• separate RNA transcript by size using electrophoresis (will smear b/c its all types of RNA)

  • capillary action (ability of a liquid to flow in narrow spaces)

• transfer to a hybridization membrane

• hybridize a labelled gene-specific probe to the RNA on the membrane to detect any RNA molecules that are homologous to the probe

  • only want the ones that stick, otherwise washed away

RNA Northern Blot FIGURE

RNA Northern Blot: Advantages vs Disadvantages

Advantages: provides transcript size, presence/absence, abundance and presence of splice variants

Disadvantages: Time consuming

RNA Dot Blot

• isolate RNA from cells

  • different tissues

  • the same tissue at different developmental times

• spot RNA onto blot

• hybridize to a labeled probe

• if target sequence is present, can detect that label

• no need to separate by size

RNA Northern vs Dot Blot

Northern Blot: - more time consuming

• provides info about presence/absence of transcript

• gives info about transcript size and presence of splice variants

Dot Blot: - simpler, faster

• provides info about presence/absence of transcript

• lacks info about transcript size or presence of splice variants

Methods for detecting Transcript Distribution: Reverse Transcriptase (RT-PCR)

• starts w/ a mRNA template

• mRNA → anneal oligo-dT primer to poly A tail → reverse transcriptase → cDNA made of all transcripts present in the tissue → use cDNA in conventional PCR reaction w/ primers specific to your transcript of interest (gene-specific primers)

• more sensitive at detecting specific RNAs than RNA blot

RT-PCR Steps

• isolate RNA from cells from different tissues or the same tissue at different developmental times

• make cDNA from mRNA using reverse transcriptase and oligo-dT primer

• use gene specific primers to amplify cDNA (if the transcript corresponding to the primers is present)

  • i.e carry out PCR on the cDNA

• run PCR reaction on a gel to observe if there is an amplified product

PCR and RT-PCR are only somewhat quantitative

• band intensity reflects how many copies of template DNA were present at the start of PCR

• fainter bands means less starting template and less overall amplification

• over the 25-40 cycles of a typical PCR, the amount of DNA product reaches a plateau that is not directly correlated w/ the amount of target DNA in the initial PCR

Solution: Quantitative (Real-time) PCR and RT-PCR (qPCR + qRT-PCR)

• include a probe w/ a reporter that fluoresces only when new DNA is synthesized (e.g SYBR green, which fluoresces when bound to dsDNA)

• amount of fluorescence measured reflects the total amount of amplified DNA present

  • measuring the amount of PCR product after every cycle

• analyze how fluorescence changes w/ PCR cycle

Quantitative (Real-time) PCR and RT-PCR (qPCR + qRT-PCR) CONTINUED

• normal PCR and RT-PCR: we visualize DNA after PCR cycles are complete

  • amplified DNA already at a plateau

• qPCR: we measure CT (threshold cycle), the number of PCR cycles it takes for detected fluorescence to be greater than threshold levels – before the plateau is reached

Model of Real-Time PCR

• legend: tenfold differences in the amount of template

• each sample hits the threshold at a different cycle number

  • reflects how much template we started w/

  • whatever hits the threshold first has more copies of template DNA

• regular PCR and RT-PCR: amplified DNA already at a plateau (not much info about starting amounts of template)

Advantages/Disadvantages of RT-PCR and qRT-PCR

Advantages: fast, sensitive

Disadvantages: no information on transcript size

• RT-PCR is crudely quantitative

• subject to artifacts (contamination)

In Situ Hybridization

• section organism/organ/tissue of interest

• hybridize a gene specific probe to a tissue section on a slide, or to whole embryo if small

• if any cells within the sectioned tissue have transcripts of the gene of interest (matches the probe), the probe will hybridize to those cells

  • detection of the probe will identify those cells

In Situ Hybridization: Advantages + Disadvantages

Advantages: provides precise information on spatial distribution of gene transcript

• can be combined w/ time (take slices of organism at diff developmental time points)

• can see where + when transcripts show

Disadvantages: difficult, time consuming, differences in tissue slice

• not as quantitative as other methods (little information on amount of transcript)

  • not as sensitive/precise as quantitative PCR

Example: Arabidopsis Floral Development

• looking at where agamous gene might be transcribed (using tissue slice of developing bud since gene has to do w/ flower development)

• imaging: Inflorescence SEM (surface structures), Inflorescence Section (transmitted light microscopy; internal structures)

• AG gene-specific probe hybridized only to cells containing AG transcript. Probe appears as blue coloration

  • denser color = more transcript

Gene Expression in WT Tissue

• gene specific probe hybridizes only to cells containing the target transcript

• probe signal is visualized as a color change

• denser color = more transcript in expected surface area

Consider:

• does the observed expression pattern match what you would expect based on the gene’s known function?

• can you estimate the abundance of transcripts in different cells or regions

Microarray

• each dot represents one probe

• each probe is specific for a different mRNA transcript

• looking at all transcripts that might be produced by cells/tissues at one time

• spot different probes onto blot → hybridize to labeled full-length RNA transcripts

  • apply long to short: hybridizing something long against a short template

Microarray vs RNA Dot Blot

RNA Dot Blot: spot RNA onto blot → hybridize to a single labeled probe → wherever the probe binds is where a particular transcript is being expressed

• doesn’t give information about size (little bit based on densities), may miss splice variants

• short to long: hybridizing a short piece of nucleic acid to a longer one

Microarray: long to short

Microarray Hybridization

• synthesize gene specific probes (oligonucleotides) for thousands of genes

• array the probes on a hybridization membrane or chip

• isolate RNA from a specific tissue/developmental time

  • need a reference sample and an experimental sample to see differences in gene expression

• make labelled cDNA from the RNA

  • reference and experimental samples labelled w/ diff fluorescent dyes

• hybridize equal amounts labelled cDNA to the array of probes and detect which probes hybridize to the population of cDNAs

Microarray Hybridization: Advantages + Disadvantages

Advantages: provides information on the amount of RNA transcript for every gene included in the array

Disadvantages: expensive, results must be repeated or verified by another technique, only a subset of genes/genome is represented on array

Past + Current Sequencing Technologies

• all based electrophoresis, one sequence per lane capillary

Next Generation Sequencing: RNAseq Background

• not based on electrophoresis

• millions to billions of sequence reactions in parallel (massively parallel sequencing)

• sequences are generally short (50-300 bp)

• cost per base pair is much lower

RNAseq Overview

• isolate RNA from tissues

• make cDNA and fragment to smaller pieces (thru RT-PCR)

  • more stable than RNA

• add PCR adaptors to fragments

  • adding short DNA sequences of known sequence to both ends of unknown cDNAs

  • all cDNA fragments now flanked by known sequences

• generate array of PCR colonies by bridge PCR (using primers against adaptors sequences)

  • i.e PCR carried out on fixed surface (membrane)

• sequence each PCR colony

• assemble PCR fragments into full sequence

Bridge PCR: Key Ideas

• randomly generated DNA fragments were ligated to adaptors in step 3

• one end of each DNA fragment is fixed to a solid surface

• surface is also coated w/ forward and reverse PCR primers that correspond to the adaptors

• bends backwards to anneal to primers on chip → duplicate DNA → now have copied piece of DNA that’s also fixed at one end

Bridge PCR

• amplification proceeds in cycles, with one end of the DNA tethered to the surface

• after several cycles, each amplified genomic fragment results in a cluster of fragments on the surface

• each colony/spot represents a different PCR product, from a different fragment

• we amplified everything to have enough fluorescence for a readout → can move onto sequencing step

Reversible Terminator Chemistry

Sequencing by Synthesis

• enzymatic extension w/ fluorescently tagged nucleotides

• the sequence of each DNA fragment (each amplified into a cluster of identical fragments) is recorded simultaneously; the newly added base for each cluster is read w/ each cycle

Cycle 1: add sequencing reagents → first base incorporated → remove unincorporated bases → detect signal → cleave block and fluorescent groups

Cycle 2-n: add sequencing reagents and repeat

Sequencing Cycles Figure

Assemble Fragments

• computers used to assemble the sequences of all the fragments into a single sequence

• see where sequence fragments are overlapping → allows assembly into longer sequence

• if you have a reference genome, you can compare sequence against reference (helps w/ assembly)

• quantitative method: the more a gene is transcribed = more sequence reads

• can get info abt alternative splicing (how many exons, how often expressed)

RNAseq: Advantages vs Disadvantages

Advantages: sensitive, quantitative, provides information on all transcripts

Disadvantages: relatively expensive, spatial information is lost, need to assemble into full sequences