L10 - Methods for Measuring Gene Expression KEATS PDF

Methods to study gene expression Natalie Prescott 5BBG0205 Molecular Basis of Gene Expression Welcome to Genetics Teaching King’s College London Welcome Department @lifeatkings to King’s College London Genetics Teaching Department @l...

Methods to study gene expression Natalie Prescott 5BBG0205 Molecular Basis of Gene Expression Welcome to Genetics Teaching King’s College London Welcome Department @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings Learning outcomes At the end of this lecture, it is expected that you will be able to: Explain the properties of RNA and how it differs from DNA Understand the challenges associated with RNA and its extraction/isolation Be aware that studying RNA is one of the foremost ways we can study gene expression Give an overview of some of the key methods used to study RNA which include: Northern analysis (blotting) Reverse Transcriptase-PCR (RT-PCR) cDNA libraries Explain how QPCR can be used to quantify gene expression and the two main approaches to this Have an overview of –omics technologies for analysing gene expression Welcome to King’s College London Genetics Teaching Department @lifeatKings  The properties of RNA Welcome to King’s College London Welcome @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings What’s the difference between RNA and DNA? https://youtu.be/JQByjprj_mA?si=3Njdb5oK3KetdzDn https://youtu.be/JQByjprj_mA?si=3Njdb5oK3KetdzDn Welcome to King’s College London Genetics Teaching Department @lifeatKings Welcome to King’s College London Genetics Teaching Department @lifeatKings DNA versus RNA: 3 key differences 1. DNA is double stranded. 2. Ribose (not deoxyribose). 3. Uracil instead of Thymine RNA is single stranded. 2. Welcome to King’s College London Genetics Teaching Department @lifeatKings RNA isolation (extraction and purification) Whole organism Cultured cells Tissue sections Pathology specimen Welcome to King’s College London Genetics Teaching Department @lifeatKings RNA is unstable, but why? Vulnerable to hydrolysis Single stranded Contains ribose Ribonucleases (RNAses) - RNA destroying enzymes - are everywhere They are found in all cells They don’t require cofactors like DNAses Humans shed them in significant quantities RESULT = High turnover rate Welcome to King’s College London Genetics Teaching Department @lifeatKings  Yes the high turnover rate of RNA is essential for the precise regulation of gene expression Welcome to King’s College London Genetics Teaching Department @lifeatKings RNA isolation Tissue Homogenization RNA Extraction Purification/ collection stabilization Precipitation RNAse Welcome to King’s College London Genetics Teaching Department @lifeatKings Acid GTC-phenol-chloroform extraction Phenol-chloroform Separates nucleic acids from proteins *GTC Denatures proteins, particularly RNAses Sodium Acetate (pH4) Partitions DNA to organic phase GTC = guanidinium thiocyanate Welcome to King’s College London Genetics Teaching Department @lifeatKings Extracted whole human RNA contains multiple types of RNA What are the main types of RNA? Welcome to King’s College London Genetics Teaching Department @lifeatKings Extracted whole human RNA contains multiple types of RNA Q: What are the three main types of RNA? A: rRNA, mRNA and tRNA, and others too. Welcome to King’s College London Genetics Teaching Department @lifeatKings Poly A+ selection of Eukaryotic mRNAs rRNA is highly abundant in cells and makes up 90% of all RNA extracted It needs to be removed if you want to study the expression of protein encoding genes (mRNAs) In Eukaryotes we can utilise the polyA tail on mRNAs to purify them from other RNAs. Oligo dT bead Used for poly A+ selection Welcome to King’s College London Genetics Teaching Department @lifeatKings Summary of properties of RNA RNA is much less stable than DNA for a good reason – it facilitates precise regulation of gene expression. Ribonucleases are everywhere and will degrade RNA so during extraction we usually inhibit these with GTC (guanidinium thiocyanate). Regardless of the source (type of tissue), mRNA only makes up a small proportion of the total extracted RNA. For these reasons we often employ a technique called poly A selection during or after extraction. Welcome to King’s College London Genetics Teaching Department @lifeatKings  Techniques used to study gene expression from RNA Welcome to King’s College London Welcome @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings Northern analysis (blotting) Named after the Southern blot (Edwin Southern) originally used for separating fragmented genomic DNA 4 11. Extracted whole RNA or polyA+ RNA is applied to a horizontal gel for electrophoresis ‘Northern’ gel/analysis 22. The resultant pattern of RNA molecules is transferred onto a nitrocellulose membrane 1 3 33. The RNA now immobilised on the membrane is incubated with a radioactive/fluorescent probe complimentary to your gene of interest 2 44. Visualisation of the probe will show if the RNA was present and its size (using a size marker/ladder) Welcome to King’s College London Genetics Teaching Department @lifeatKings ‘Northern’ analysis comparing the gel to the blot UV image of Ethidium bromide stain X-ray image of Northern blot using of total RNA ‘Northern’ gel specific staining with radiolabelled probe Welcome to King’s College London Genetics Teaching Department @lifeatKings Northern blot example In 1989, Riordan et al used a Northern blot to confirm the expression pattern of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Welcome to King’s College London Genetics Teaching Department @lifeatKings Summary of Northern analysis Gel electrophoresis of whole or total RNA extracted from a tissue or cell is usually referred to as a ‘Northern’. Transferring the separated RNA molecules from a ‘Northern’ gel to a nitrocellulose membrane makes it easier to probe for a specific transcript of interest. Northern blotting can be useful for showing the relative amount and sizes (length) of specific transcripts (RNAs). But it is a challenging technique which is time-consuming and requires a lot of input RNA sample. Welcome to King’s College London Genetics Teaching Department @lifeatKings  Reverse Transcription-PCR (RT-PCR) Welcome to King’s College London Welcome @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings The polymerase chain reaction – a recap A method for copying DNA Requirements: DNA template oligonucleotide primers DNA polymerase enzyme (e.g. Taq polymerase) Nucleotides (A, C, G, T) https://www.youtube.com/watch?v=2KoLnIwoZKU Welcome to King’s College London Genetics Teaching Department @lifeatKings The phases of PCR 11. Lag – when PCR is initiating but undetectable 4. plateaux phase 2 2. Exponential – PCR Amount of PCR product components are in excess. Product doubling occurs. 3. linear phase 3 3. Linear – efficiency declines Threshold of detection with each cycle 1. Lag phase/initiation 2. Exponential phase 44. Plateaux – PCR components have run out PCR cycle number Welcome to King’s College London Genetics Teaching Department @lifeatKings Reverse Transcription PCR (RT-PCR) RT-PCR is an adaptation to to PCR …with some key differences The template that is copied is mRNA mRNA cannot be amplified directly as no enzyme exists for it Therefore, we must first use reverse transcriptase to convert RNA to DNA Welcome to King’s College London Genetics Teaching Department @lifeatKings Reverse transcriptase makes complimentary DNA (cDNA) from RNA cDNA The enzyme originates in RNA RNA viruses called retroviruses Retroviruses use the enzyme to RT reverse-transcribe their RNA genomes into DNA RNA This allows them to be integrated into the host genome and be RNA replicated along with it Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: The viral reverse transcriptase enzyme catalyzes the reverse transcription reaction. Why is it called reverse transcription? (A) The enzyme can transcribe genes in the reverse direction (3' -> 5') to normal (which is 5' -> 3’) - INCORRECT (B) The enzyme catalyses transcription from the reverse strand of the DNA - INCORRECT (C) The enzyme can catalyze the reverse process of normal cellular transcription, i.e. RNA -> DNA - CORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings RT-PCR is a two-step process Step 1: Reverse transcription (RT). The reverse transcriptase enzyme is used to generate the first strand which is a DNA copy of the RNA Step 2: PCR Taq polymerase is added, and PCR can proceed in the traditional way Welcome to King’s College London Genetics Teaching Department @lifeatKings Priming the first step (RT) BOTH RANDOM Random hexamers Oligo dT Gene specific primers A mixture of oligonucleotides Single stranded sequence Oligonucleotide designed to be Representing all possible 12-18 deoxythymidines perfectly complimentary to a hexamer sequences [TTTTTTTTTTTT] gene [NNNNNN] Will hybridise to poly A tail of Requires sequence knowledge Will randomly hybridise to any mRNAs sequence RT copies ‘all’ RNAs RT copies all RNAs with poly A RT copies one gene e.g. mRNA 5’- Coding part of gene Poly A -3’ 5’- Coding part of gene Poly A -3’ 5’- Coding part of gene Poly A -3’ RNA RNA RNA Welcome to King’s College London Genetics Teaching Department @lifeatKings The two-steps of RT-PCR can either be done simultaneously or sequentially One step (simultaneous) RT PCR Two step RT PCR All reagents are added at the RT reaction occurs separately beginning from PCR Option to stop here Both reactions happen and store sample Can use random priming to simultaneously make the first strand of cDNA Requires a specific primer from the outset ADVANTAGE: can stop after 1st ADVANTAGE: Quicker than two - step and store cDNA for use in step https://goldbio.com/goldbios-pcr-overview other PCRs later Welcome to King’s College London Genetics Teaching Department @lifeatKings Example of RT-PCR: identifying different splice isoforms of the TUCAN/CARD8 gene A DNA change that causes a nonsense mutation (p.10 Cys>STOP) within exon 5 of TUCAN/CARD8 gene had been associated with inflammatory bowel disease The mutation introduces a premature termination codon (STOP) into the main 48kDa isoform of the gene (aka T48) at the 10 th amino acid residue Bagnall, EJHG 2008. https://www.nature.com/articles/5201996 Welcome to King’s College London Genetics Teaching Department @lifeatKings Example of RT-PCR of TUCAN We used gene specific RT-PCR on mRNA from blood of individuals with and without the mutation and other reference tissues to show there were at least 3 more isoforms of TUCAN Welcome to King’s College London Genetics Teaching Department @lifeatKings DNA change does not cause a STOP mutation in new TUCAN isoforms Bioinformatics and cDNA sequencing showed that the different isoforms were due to inclusion of a new exon (4a) or skipping exon 6. The new T60 and T47 isoforms changed the open reading frame of the transcript in such a way that the mutation did not result in a STOP Amplification of individual isoforms using isoform specific primers Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: What is the primary purpose of RT-PCR? A. To amplify DNA sequences - INCORRECT B. To synthesise DNA from an RNA template - CORRECT C. To denature DNA - INCORRECT D. To detect protein expression -INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: I am about to set up an RT-PCR reaction. I have my RNA template, some reverse transcriptase, an RT primer (oligo dT) and a PCR machine. What else do I need? A. Deoxyribonucleotide triphosphates – CORRECT B. Ribonucleotide triphosphates – INCORRECT C. Amino acids – INCORRECT D. Base pairs - INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: Which of the following is a characteristic of using random priming in reverse transcription PCR (RT-PCR)? A. Specific primers are designed to target a single gene of interest. -INCORRECT B. It does not require a separate reverse transcription step before PCR. -INCORRECT C. Random primers can initiate cDNA synthesis at multiple sites and/or multiple RNAs. - CORRECT D. It is primarily used for DNA sequencing applications.-INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Summary of RT-PCR RNAs cannot be amplified directly so we have harnessed the viral enzyme Reverse Transcriptase that can make a DNA copy of RNA. We call this copy cDNA (complementary DNA) RT-PCR is a two-step process that uses reverse transcriptase and then a DNA polymerase; but, depending on the need, the technique can be adapted so that both steps are either done simultaneously (‘one-step’) or sequentially (two-step) Random primers such as hexamers or oligo dT will amplify multiple RNAs simultaneously but specific primers can be designed to target one gene or one isoform of a gene. Once RNA is converted to cDNA it is much more stable and easier to store long term Welcome to King’s College London Genetics Teaching Department @lifeatKings  Quantitative Real Time PCR Welcome to King’s College London Welcome @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings  Quantitative real time PCR (QPCR) 1st cycle 2nd cycle 3rd cycle 4th cycle completion/end Traditional PCR Real time QPCR Welcome to King’s College London Genetics Teaching Department @lifeatKings How to measure the reaction in real time 1. Label the accumulating PCR product (DNA) 2. Measure the fluorescence Fluorescent dye (SYBR Green) Binds all double stranded DNA OR Fluorescent probe & quencher (TaqMan) Q Target specific gene Q PCR machine = PCR machine plus camera Welcome to King’s College London Genetics Teaching Department @lifeatKings  SYBR Green I An asymmetrical cyanine dye used in molecular biology Preferentially binds (intercalates with) double stranded DNA (dsDNA) Binding of dsDNA causes a dramatic increase in fluorescence >1,000 fold Welcome to King’s College London Genetics Teaching Department @lifeatKings Taqman (5’ nuclease) assay Taqman probes are dual labelled with a fluorescent dye and a quencher They are designed to specifically hybridize a chosen gene’s PCR product as it accumulates The quencher prevents the dye from fluorescing whilst the probe is intact During each cycle of the PCR the bound probe is cleaved by the 5’ nuclease activity of Taq polymerase and dye is released allowing it to fluoresce. As PCR product increases, so does fluorescence. Welcome to King’s College London Genetics Teaching Department @lifeatKings Quantitative real time PCR (QPCR) https://youtu.be/1kvy17ugI4w?si=zvfZfkUDNMF4x5q2 Welcome to King’s College London Genetics Teaching Department @lifeatKings Typical real time PCR plot The point at which the amplification plot crosses the threshold is called the Ct value (cycle threshold). The threshold is set so that we can measure Ct during the exponential phase. Fluorescence Here Ct = 28.5 Threshold PCR cycle number Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: Which statement/s below is true when we describe what is happening during the exponential phase of PCR A. PCR components are running out - FALSE B. PCR product is doubling with every cycle - TRUE C. The amount of PCR product is directly related to the amount of starting product (template) - TRUE Welcome to King’s College London Genetics Teaching Department @lifeatKings Typical real time PCR plot The point at which the amplification plot crosses the threshold is called the Ct value (cycle threshold). The threshold is set so that we can measure Ct during the exponential phase. Fluorescence Here Ct = 28.5 Threshold By comparing Ct values to another sample (comparator), we can calculate a relative expression. Comparator sample will usually be a sample that expresses the gene PCR cycle number at a low level Welcome to King’s College London Genetics Teaching Department @lifeatKings Relative quantitation uses the ΔCt method Sample A Ct = 29 (rounded up) Sample B Ct = 24 Difference (ΔCt) = 5 Sample B Sample B contains 25 (32) times more of the gene of interest than sample 1 Fluorescence Sample A It’s 25 (2 to the power of 5) because of sample doubling every cycle Threshold RQ[B] = 32 RQ[A] = 1 In practice we would also use a housekeeping gene to control for PCR cycle number technical errors such as RNA amount. Welcome to King’s College London Genetics Teaching Department @lifeatKings What is a housekeeping gene? A gene that is constitutively active Required for basic functions Expressed at a similar level in all tissues/cells Unaffected by the treatment of your test samples Acts as an endogenous (within sample) control Controls for biases in sample collection or extraction Some well-known housekeeping genes GAPDH – glyceraldehyde-3-phosphate dehydrogenase ACTB – beta actin B2M – beta 3 microglobulin BTUB – beta tubulin Welcome to King’s College London Genetics Teaching Department @lifeatKings Example: QPCR to examine IRGM expression in individuals with a promotor variant Identify CD patients with and The IRGM gene region has been associated without the IRGM promotor with Crohn’s disease by genome wide deletion association study (GWAS). Collect RNA samples from IRGM is involved in the immune response. immune cells (blood lymphocytes) A genetic variation (deletion) in the promotor of the gene is responsible for the association Carry out qPCR of IRGM and the with Crohn’s. housekeeping gene GAPDH Does this deletion affect the expression of the Also include a comparator gene? Δ sample (placenta) Prescott et al (2010) Hum Mol Gen https://doi.org/10.1093/hmg/ddq041 Welcome to King’s College London Genetics Teaching Department @lifeatKings The delta delta Ct method for calculating Relative Quantification in QPCR Δ is the Greek symbol for delta Test gene (IRGM) Housekeeping gene DISCLAIMER! (GAPDH) DISCLAIMER Test sample A for your information. Just B (LymphocyteYOU RNA)DO NOT NEED TO REMEMBER THIS You do not need to memorise this equation Comparator sample C EQUATION D (placental RNA) Δ Ct [lymphocyte] = A-B 𝑅𝑄 = 2−ΔΔ 𝐶𝑡 Δ Ct [placenta] = C-D Δ Δ Ct = (Δ Ct [lymphocyte] )-(Δ Ct [placenta] ) Welcome to King’s College London Genetics Teaching Department @lifeatKings QPCR via RQ method in lymphocytes from Crohn’s disease patients with a promotor variant in IRGM gene 18 Normal Heterozygous (+/-) Homozygous (+/+) 16 (no variant) 14 Relative Quantiity [IRGM] 12 mean RQ = 13 mean RQ = 3 mean RQ = 1 10 8 6 4 2 0 IBH0478 IBH0692 IBH0676 IBH0273 IBH0545 IBH0628 IBH0912 IBH0830 IBH0216 IBH0272 IBH0212 IBH0245 GKT0540 IBH0226 IBH0365 Placenta Sample Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: What can you observe about the relative quantity of IRGM expression in the 'normal' samples compared to the low expressing placental control. A. On average there is a 13-fold greater expression of IRGM in normal lymphocytes than the placental control. - CORRECT B. On average there are 13 more copies of IRGM expressed in normal lymphocytes than the placental control. - INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: What might be causing difference observed between the 'normal' samples (without the IRGM promotor variant) and the homozygous samples (with two copies of the promotor variant) A. There was much more total RNA in the 'normal' samples – UNLIKELY (we used GAPDH endogenous/housekeeping gene to control for this) B. The promotor variant is affecting the expression of IRGM – YES this is possible (but would need further experiments to confirm) Welcome to King’s College London Genetics Teaching Department @lifeatKings The absolute quantification method uses a standard curve instead of a housekeeping gene Standard curve A dilution series of a predefined amount of a PCR product of the gene/cDNA e.g. serial dilution from 108 to 101 copies Each of the sample dilutions are used in a separate real time PCR reaction The Ct value is calculated for each sample Welcome to King’s College London Genetics Teaching Department @lifeatKings The standard curve is used to infer the ‘Absolute Quantity’ of an unknown sample via Ct value Ct values from the dilution series are plotted on a graph (standard curve) This can be used to estimate the amount of an unknown sample using it’s Ct value from QPCR In practice the standard curve samples and the unknown samples are usually run at the same time on the same machine Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: Which of the statements below do you think are advantages of the absolute QPCR method (there may be more than one) A. You don't need to co-amplify a housekeeping gene for every sample during the reaction – ADVANTAGE B. You need to have a previously quantified cDNA of the target gene to be able to set up a standard curve – DISADVANTAGE (in my opinion!) C. You get an accurate estimate of the true quantity of the target mRNA rather than a relative quantity (fold difference) - ADVANTAGE Welcome to King’s College London Genetics Teaching Department @lifeatKings Applications of QPCR MUTATION DETECTION DNA (GENE) COPY NUMBER BACTERIAL LOAD mRNA QUANTIFICATION (GENE EXPRESSION) qPCR VIRAL LOAD Quantitative Highly sensitive Welcome to King’s College London Genetics Teaching Department @lifeatKings QPCR example in a clinical setting: Acute Promyelocytic Leukaemia PML is caused by a chromosomal translocation in white blood cells which creates a fusion gene PML/RARA Welcome to King’s College London Genetics Teaching Department @lifeatKings QPCR using primers to detect expression of the fusion gene can track treatment response in PML Primers used for qPCR of PML/RARA transcripts qPCR result for a group of patients after each round of chemotherapy Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: What is the main advantage of quantitative PCR (QPCR) over conventional PCR A. It doesn't require DNA or RNA templates. - INCORRECT B. It can detect the presence of specific genes. – INCORRECT (conventional PCR can do this too) C. It provides data on the amount of target (input) DNA or RNA. - CORRECT D. It is more reproducible - INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: In QPCR, which component is commonly used as an internal reference for data normalization? A. Sybr Green I - INCORRECT B. Random hexamers - INCORRECT C. Taqman probe - INCORRECT D. Housekeeping gene - CORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: What is the purpose of a standard curve in QPCR experiments? A. To calculate the number of PCR cycles needed for amplification. - INCORRECT B. To determine the efficiency of the DNA polymerase enzyme. INCORRECT C. To establish a relationship between cycle threshold (Ct) values and target DNA or RNA concentration. - CORRECT D. To identify specific mutations in the target DNA sequence. - INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Summary of Quantitative PCR (QPCR) QPCR is like regular PCR, but you follow the reaction in real time It is quantitative because we can identify the phase of PCR when the amount of product is directly related to the amount of starting material/template (exponential phase) QPCR has many applications, one of these is for detecting & quantifying the expression level of a gene There are two main methods of detection SYBR green and Taqman probes There are two main quantification methods: Relative Quantification - achieved by comparing target sample to a comparator sample and/or housekeeping gene. Absolute Quantification - by comparing to a standard curve derived from a serial dilution Welcome to King’s College London Genetics Teaching Department @lifeatKings  Techniques used to study the transcriptome Welcome to King’s College London Welcome @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings The eukaryotic transcriptome Genome DNA transcription Coding RNA Non-coding RNA Transcriptome mRNA tRNA, rRNA, miRNA, lncRNA translation Proteome Protein Welcome to King’s College London Genetics Teaching Department @lifeatKings Facts about the transcriptome Although single stranded RNA can form secondary structures with itself The transcriptome is constantly state of ‘turn-over’ through RNA synthesis and degradation Therefore, transcriptome can change in response to : Development and differentiation Extracellular/intracellular signals Altered environment/biochemical properties of the cell The transcriptome is not synthesised de novo During cell division, every cell receives part of the parent cells transcriptome At any given time, the transcriptome is like a snapshot of a cell’s physiology Molecular biology of the cell 6th edition Welcome to King’s College London Genetics Teaching Department @lifeatKings Example techniques for studying the transcriptome cDNA libraries Subtractive screening Gene expression microarrays High throughput/massively parallel RNA-sequencing Welcome to King’s College London Genetics Teaching Department @lifeatKings What is a cDNA library? A collection of cDNAs from a cell or tissue of interest cDNAs are often inserted into a vector such as bacterial plasmid, for easy amplification and storage. The library comprises a proportion of the transcriptome from the tissue from which it was collected and thus represents the genes that were being expressed at that time It can be amplified when needed by allowing the bacteria to multiply (grow) Much more stable than storing the RNA Welcome to King’s College London Genetics Teaching Department @lifeatKings Making a cDNA library 1 1. 1 Isolate mRNA and convert to cDNA 2. 2 Ligate cDNA into RE digested vector (plasmid) 3. 3 Transform bacteria with ligated vectors and select for those with a cDNA insert (cloning) 2 44. Amplify library by growing bacteria to form colonies of individual clones 3 Each bacterial clone/colony contains one of the genes being expressed in that tissue In practice you need enough clones to represent a ‘good’ selection of genes (can be >10,000) 4 Welcome to King’s College London Genetics Teaching Department @lifeatKings cDNA libraries have many applications… A Dot Blot of cDNAs immobilised on nitrocellulose Study genes expressed in a tissue Discovery of novel genes Study of alternate splicing Compare genes that are expressed in : Different cells resistant plant normal plant Different tissues cDNA clones from Cotton plant libraries hybridised with genes (cDNAs) identified as being differentially expressed in plants At different stages/under that are resistant to fungal infection. different conditions The differentially expressed genes were identified via subtractive screening. Welcome to King’s College London Genetics Teaching Department @lifeatKings Subtractive library screening: an early method for analysis of differential gene expression (DGE) Allows detection of genes that are differently expressed between two tissues/states cDNAs from tissue of interest have adapters added (small known sequences) Then mixed with cDNAs from control tissue and similar/same genes are subtracted Early example of differential gene expression analysis (DGE) Welcome to King’s College London Genetics Teaching Department @lifeatKings Subtractive library screening: an early method for analysis of differential gene expression (DGE) Allows detection of genes that are differently expressed between two tissues/states cDNAs from tissue of interest have adapters added (small known sequences) Then mixed with cDNAs from control tissue and similar/same genes are subtracted Early example of differential gene expression analysis (DGE) Amplify unique genes e.g. by PCR Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: This figure shows 12 unknown human cDNA that have been hybridised to a dot blot of cDNA libraries from 3 different mouse tissues. Which is the best control cDNA? A: on next slide Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: This figure shows 12 unknown human cDNA that have been hybridised to a dot blot of cDNA libraries from 3 different mouse tissues. Which is the best control cDNA? A: tRNA would make a good control as it appears to be expressed at a similar level in all tissues Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: The test cDNAs were made from RNA extracted from tumour cells from a patient. What tissue is the tumour most likley to have occurred in? A. Liver B. Kidney C. Brain Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: The test cDNAs were made from RNA extracted from tumour cells from a patient. What tissue is the tumour most likley to have occurred in? A. Liver - CORRECT B. Kidney - INCORRECT C. Brain - INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: These are dot blots of normal Cotton plant cDNA library that have been hybridised with a subtracted library from a cotton plant that is resistant to fungal infection (left). Which is true of the circled cDNAs? A. They represent genes upregulated in fungal resistant cotton. B. The represent genes downregulated in fungal resistant cotton. Answer on next slide Welcome to King’s College London Genetics Teaching Department @lifeatKings Q: These are dot blots of normal Cotton plant cDNA library that have been hybridised with a subtracted library from a cotton plant that is resistant to fungal infection (left). Which is true of the circled cDNAs? A. They represent genes upregulated in fungal resistant cotton - CORRECT B. The represent genes downregulated in fungal resistant cotton. - INCORRECT Welcome to King’s College London Genetics Teaching Department @lifeatKings Summary of cDNA libraries A cDNA library is the collection of cDNAs made from the mRNA extracted from a cell, tissue or organism of interest. It is much more stable than storing mRNA. The library represents a proportion of the transcriptome at the time of sampling. One of the ways these libraries can be propagated (amplified) is via bacterial cloning using plasmid vectors. cDNA libraries have several applications that allow the comparison of expression of a many genes between two samples (differential gene expression) Depending on the cloning strategy the cDNA library will usually only represent the most highly expressed genes. Welcome to King’s College London Genetics Teaching Department @lifeatKings  Large-scale genomics methods for measuring gene expression (transcriptomics) Welcome to King’s College London Welcome @lifeatkings to King’s College London Genetics Teaching Department @lifeatKings Two main contenders Next generation sequencing: Gene expression microarrays RNA-sequencing Welcome to King’s College London Genetics Teaching Department @lifeatKings Gene expression microarrays – Lecture 12 Thousands of DNA probes are designed to represent all known genes in an organism The probes are fixed to a solid surface (chip) and their positions are precisely defined cDNA library can be applied to the array Any cDNA that hybridises to a probe on the array will cause a fluorescent signal Welcome to King’s College London Genetics Teaching Department @lifeatKings RNA sequencing - Lecture 13 Samples of interest Isolate RNAs Library preparation fragment, generate cDNA, normal cells mutated cells add oligo tags/linkers All cDNAs then undergo high throughput sequencing https://youtu.be/fCd6B5HRaZ8 https://youtu.be/fCd6B5HRaZ8 Welcome to King’s College London Genetics Teaching Department @lifeatKings  Any questions? Welcome to Genetics Teaching King’s College London Department Genetics Teaching Department Welcome @lifeatkings to King’s College London @lifeatKings