Gene expression detection methods RNA Dr. Ingrid Müller - AG von Laer 1 TRANSCRIPTIONAL CONTROL REGULATES DIFFERENTIATION Four different human cells - same genes, different structures and functions due to differential gene expression 2 THE CENTRAL DOGMA • Flow of Information: DNA Replication Transcription Translation A B Cells in all living organisms are continually activating or deactivating genes through gene expression, which contain the information required for producing proteins through proteins synthesis. When a particular protein is required by the cell, the gene coding for that protein is activated. The first stage in producing a protein involves the production of an RNA copy of the gene's DNA sequence. This RNA copy is the messenger RNA. The amount of mRNA produced correlates with the amount of protein eventually synthesised and measuring the amount of a particular mRNA produced by a given cell or tissue is often easier than measuring the amount of the final protein. Levels in gene activation may vary between cancerogenic and healthy cells. 3 MOLECULAR METHODS TOOL BOX I. Analysis II. Overexpression III. Inhibition •Western Blot •(adding proteins to in •pharmacological inhibitors, •Proteomics vitro reactions) •dominant-negative Protein •immuno- proteins, histochemisty • protein depletion using •protein chimera antibodies •Electrophoresis •microinjection •RNAi & •Northern Blot •siRNA •RNAse protection •Morpholinos RNA assays •Microarrays •RT-PCR •RNA in-situ •Reporter genes •(microinjection) •Knock-out •Electroporation, •Integrational mutagenesis DNA (gene) •lipofection, •Classic genetics •transgenics 4 RNA METHODS: ELECTROPHORESIS 5 PURIFICATION OF MESSENGER RNA USING OLIGO DT COLUMNS Total cellular RNA; apply at room temperature to Break open anneal polyA tail to oligo(dT) cells in the presence of RNAse Oligo(dT) inhibitors attached to AAAA.. cellulose TTTT TTTT polyA binds to 65oC Isolate RNA oligo(dT) on column } non-polyA AAAAA.. RNA flows through polyA mRNA elutes at high temperature 6 RNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS 7 RNA METHODS: ELECTROPHORESIS Detection using SYBR Green II: staining of ss nucleic fragments • Denaturing of RNA to break up secondary structures • TAE / TBE running buffer • Agarose / Polyacrylamide gels 8 NORTHERN BLOTS • Isolated mRNA separated on gel according to size • mRNA transferred to a membrane and hybridized with small number (1-5) of radioactively labeled DNA/RNA probes (35S or 32P) • Probe corresponds to gene of interest • Target RNA is spatially fixed and the labeled probe is in solution • Reverse northern blot: Probe: isolated mRNA Substrate (fixed to membrane): DNA/RNA fragments 9 NORTHERN BLOT 10 NORTHERN BLOT 11 NORTHERN BLOT control sample target gene 10x internal control gene actin, GAPDH, RPLP0 etc. 2x Corrected fold increase = 10/2 = 5 Ratio target gene in experimental/control = fold change in target gene fold change in reference gene How can amounts of RNA be quantified? This slide shows a virtual Northern with two lanes, one with RNA from control cells, the other with RNA from the experimental sample (eg drug treated cells). Let’s say that there is 10x the amount of signal in the experimental sample compared to the control sample for the target gene. This could mean expression of the gene has increased 10- fold, or it could mean that there is 10x as much RNA in the expt lane. To check for this one usually does a so-called ‘loading control’ in which the blot is probed for expression of a gene which does not change (e.g. actin, GAPDH, cyclophilin, RPLP0 mRNAs; ribosomaL RNA). In this case, let’s say that the loading control shows that there is twice as much RNA in the expt lane. Thus the real change in the target gene is 10/2 =5 fold. We can express this in a more general fashion: ratio targ et g ene ( experim ental/control) = fold chang e in targ et g ene ( expt/control) fold chang e in reference g ene ( expt/control) 12 NORTHERN BLOT Measure relative expression levels of mRNA 1. mRNA isolation and purification 2. electrophorese on a gel 3. The gel is probed by hybridizing with a labeled clone for the gene under study. northern blotting of human tissues 13 RNA METHODS: NORTHERN PRO’S AND CON’S Pros – Established and widely accepted method for single mRNA species detection – Newer techniques can be done without radiation (fluorescence) Cons – Same semi-quantitative limitations as seen in SDS- PAGE/Western Blots for protein – Time consuming – Low throughput Perform 35,000 Northerns to monitor expression of all genes!!! 14 MICROARRAYS 15 FINGERPRINTS OF GENE EXPRESSION ⇒ MICROARRAYS Normal Cell Cancer Cell 16 MICROARRAY Collection of microscopic DNA spots attached to a solid surface (glass, plastic, silicon chip) forming an array for the purpose of expression profiling, monitoring expression levels for thousands of genes simultaneously Applications: Analysis of expression patterns in: – Tissues – Disease states Sub-typing complex genetic disease e.g. cancer 17 DNA ARRAY TECHNOLOGY Spot Density Array Type Probe Target Labeling (per cm 2 ) Nylon Macroarrays < 100 cDNA RNA Radioactive Nylon Microarrays < 5000 cDNA mRNA Radioactive/Flourescent Glass Microarrays < 10,000 cDNA mRNA Flourescent Oligonucleotide Chips <250,000 oligo's mRNA Flourescent Fabrication Printing using fine-pointed pins on glass slide Photolithography 18 PHYSICAL SPOTTING 19 PHOTOLITHOGRAPHIC SPOTTING 20 TWO POPULAR MICROARRAYING PLATFORMS Spotted microarrays Commercially available Oligonucleotide microarrays Probes: Affymetrix “Gene Chips” Synthesized oligos (70 mer) Probes: cDNA Oligos (25 mer),represent gene fragments Small PCR products (500-1,000bp) Produced by photolithography on silica Corresponding to mRNAs matrix >10,000 probes 500,000 probes 21 SPOTTED MICROARRAYS tumor normal Pro Mixing Only one chip needed per experiment Con Absolute gene expression levels cannot be measured 22 GENECHIPS® BY AFFYMETRIX cDNA 23 GENECHIPS® BY AFFYMETRIX Single nucleotide polymorphism (SNP): responsible for genetic variation and the source of susceptibility to genetically caused diseases SNP microarrays: particular type of DNA microarrays used to identify genetic variation in individuals and across populations Applications: Forensics Measurement of genetic predisposition to disease Profiling somatic mutations in cancer 24 MICROARRAY “Heat map” Here we can see an annotated close-up of an affymetrix chip, with the regions relating to several genes highlighted. 25 GENECHIPS® BY AFFYMETRIX 26 THE PROBLEM NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION THE SOLUTION • PCR: - most sensitive - can discriminate closely related mRNAs - technically simple - but difficult to get truly quantitative results using conventional PCR Real time PCR was developed because of the need to quantitate differences in mRNA expression. PCR methods are particularly valuable when amounts of RNA are low since the fact that they involve an amplification step means they are more sensitive. 27 RT-PCR REVERSE TRANSCRIPTASE-PCR • RNA containing virus – PCR doesn’t work on RNA templates Extract RNA from cells – RT PCR RNA • make cDNA copy of RNA sequence first • PCR the cDNA copy of RNA PCR Can observe very low levels of expression Requires very small amounts of mRNA Have to design multiple custom primers for each gene 28 RT-PCR Measures relative expression of mRNA 1. Isolate and purify mRNA 2. reverse transcription 3. PCR amplification 4. run on gel First, the mRNA’s are isolated and purified. Next, the mRNA is reverse transcribed, possibly using a gene-specific primer. Recall that transcription normally makes an RNA copy from a DNA template. This is reverse transcription, as it is making a DNA copy from an RNA template. Next, standard PCR is used to amplify the number of copies of the transcript under study. Finally, the resulting product is run out on a gel and probed. 29 PCR Conventional 100 PCR t n u o m A t c u d o r P 0 Cycle number 35 Regular PCR involves performing the reaction and electrophoresing the final product – not reflective of starting amount 30 WHAT’S WRONG WITH AGAROSE GELS? * Poor precision * Low sensitivity * Short dynamic range < 2 logs * Low resolution * Non-automated * Size-based discrimination only * Results are not expressed as numbers * Ethidium bromide staining is not very quantitative 31 REAL TIME PCR Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection • kinetic approach • early stages • while still linear Real time PCR is a kinetic approach, where you look at the reaction in the early stages while it is still linear. There are many real time machines available. This is the one we use (the BioRad Icycler IQ real time PCR instrument). The lid slides back and then we put samples in a 96-well plate format inside, so one can look at a lot of samples simultaneously. The machine contains a sensitive camera which monitors the fluorescence in each well of the 96-well plate at frequent intervals during the PCR reaction. In our case, as DNA is synthesized, more SYBR green will bind and the fluorescence will increase. 32 REAL-TIME qPCR • What is real-time quantitative PCR? A PCR-based method to measure the number of copies of a particular DNA fragment in a given sample - Amplification products are labeled by a DNA binding dye or probe chemistry that emit fluorescent signal when excited. -The signal strength of the emitted light is directly proportional to the amount of PCR product in the reaction