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An introduction to qPCR BCH452 Spring 2014 So far in BCH452 you have studied • • • • Carbohydrates: Glucose Proteins: ADH and LDH DNA: Plant DNA and viral DNA What is missing? – This week we will study RNA (indirectly). Methods for quantifying DNA and RNA—Blotting Methods for quantifying DNA and RNA—Microarrays Quantifying DNA/RNA by qPCR Reminder: PCR What is qPCR • “quantitative Polymerase Chain Reaction” • A method that allows to follow in real time (that is why is also called Real-Time PCR) the amplification of a target. • The target can be nucleic acids (RNA or DNA). • Taq polymerase can only synthesize DNA, so how do we study RNA using qPCR? Reverse Transcription • mRNA can be copied to complementary DNA sequence (cDNA) using reverse transcriptase—a DNA polymerase that uses ssRNA as template. • Processed mRNA will match protein coding sequence while unprocessed (nuclear) mRNA will contain intron sequences. Uses of qPCR • Precise quantitation of DNA or RNA in samples • Estimation of gene number • Gene expression studies by quantification of messenger RNA Central Dogma of Molecular Biology: From DNA to RNA to protein Principle of gene expression: From DNA to RNA to protein What are we doing today? • We are using qPCR as a way to study RNA, in this particular case messenger RNA (mRNA). • We will test the gene expression response of a plant to a geminivirus infection. • This can be applied to any gene expression analysis. Reminder: PCR Different chemistries involved to obtain quantitative signal energy qPCR thermalcyclers detect fluorescence fluorophore FRET acceptor fluorophore + quencher in proximity FRET donor TaqMan Molecular beacons Molecular probes SYBRGreen We are using relative expression analysis but absolute expression analysis is possible with qPCR ΔΔCt method test 2 reference -[(Ctinf-Ctmock)-(Ctinf-Ctmock)] Cabbage Leaf Curl Virus (CaLCuV) • Using qPCR to detect changes in gene expression. – Mock tissue (uninfected) – Infected tissue Arabidopsis thaliana • Looking at genes involved in pathogen response. • cDNA prepared from RNA purified from Arabidopsis plants – Reverse transcription using polyT primer and NTPs. Arabidopsis infected with CaLCuV Pathogen response in arabidopsis Salicylic acid Jasmonic acid Ethylene Genes of interest for today • • • • • • • • PR1- Pathogenesis-related NPR1- Not expressing PR-1 WRKY53- Transcription factor involved in pathogen response FAD8- Fatty-acid desaturase, involved in the production of jasmonate (pathogen response) Invertase- Enzyme known to be involved in the pathogen response Catalase- Enzyme known to be involved in the pathogen response CAB-AB- Protein that takes part in the Chlorophyll complex UCE- Ubiquitin conjugating enzyme. Control gene. It has been reported it doesn’t change during infection. Prior to doing the qPCR • Total RNA from mock and infected plants was purified • It was treated with DNAase to get rid of all genomic DNA (will also amplify with primers) • cDNA was produced and diluted to 50ng/µl Preparing a qPCR reaction • Forward and reverse primers (oligos) for each gene of interest • cDNA of control and treated samples (mock and infected, respectively) • SYBRGreen PCR mix • Thermocycler with fluorescence measuring capabilities Each team (7 teams total) • Will prepare mixes with oligos and cDNA for a SINGLE gene (for both mock and infected). 35 μL primer mix add to cDNA 20 μL primer/cDNA In each well 35 μL primer mix add to cDNA 20 μL primer/cDNA In each well Each team • The assay will be tested in triplicate (three technical replicates). M M M I I I Each team The whole section will analyze the data, the same way as it was for the pre-lab example. – Calculate ΔCt for each gene. – Calculate ΔΔCt for 7 test genes relative to UCE. – Calculate log10(ΔΔCt) for 7 test genes to determine increase or decrease in expression. – Make bar chart of log10(ΔΔCt) for each gene – Construct table with “call” if gene was upregulated, downregulated or showed no change Analysis requirements 2.5 Relative gene expression log10 of relative gene expresion 2 1.5 Gene A B C D E F G UCE 1 0.5 0 -0.5 -1 -1.5 Gene Value 1.828745739 2.04179718 0.666738198 1.999855536 - 0.450673805 0.509327854 - 1.195371388 0 Call up regulated up regulated unaffected up regulated unaffected unaffected down regulated control gene