Understanding Real-Time PCR and its Applications
Real-Time PCR, also known as quantitative PCR, is a specialized technique used in molecular biology for gene expression analysis, medical research, disease diagnosis, viral quantification, and more. It enables the real-time visualization and measurement of DNA sequences in a sample, playing a crucial role in various fields such as genetic profiling, disease management, and monitoring viral loads for treatment decisions. This presentation covers the fundamentals of Real-Time PCR, its applications in gene expression analysis and disease management through examples like BRCA1 expression profiling and HIV treatment monitoring.
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Presentation Transcript
Real-Time PCR (RT PCR) aka Real-Time quantitative PCR (RT qPCR) DAVID A. PALMER, PH.D. TECHNICAL SUPPORT, BIO-RAD LABORATORIES ADJUNCT PROFESSOR, CONTRA COSTA COLLEGE MINOR MODIFICATIONS BY J. MATTSSON AND K. FITZPATRICK
OBJECTIVES THIS PRESENTATION WILL COVER THE FOLLOWING TOPICS: WHAT IS REAL-TIME PCR USED FOR? HOW DOES REAL-TIME PCR WORK? WHAT INSTRUMENTS ARE USED? WHAT DOES REAL-TIME DATA LOOK LIKE?
PART 1: WHAT IS REAL-TIME PCR AND WHAT IS IT USED FOR?
THE POLYMERASE CHAIN REACTION (PCR) IS A PROCESS FOR THE AMPLIFICATION OF SPECIFIC FRAGMENTS OF DNA. REAL-TIME PCR A SPECIALIZED TECHNIQUE THAT ALLOWS A PCR REACTION TO BE VISUALIZED IN REAL TIME AS THE REACTION PROGRESSES. WHAT IS REAL-TIME PCR? AS WE WILL SEE, REAL-TIME PCR ALLOWS US TO MEASURE MINUTE AMOUNTS OF DNA SEQUENCES IN A SAMPLE!
REAL-TIME PCR HAS BECOME A CORNERSTONE OF MOLECULAR BIOLOGY: GENE EXPRESSION ANALYSIS MEDICAL RESEARCH BASIC RESEARCH WHAT IS REAL-TIME PCR USED FOR? DISEASE DIAGNOSIS AND MANAGEMENT VIRAL QUANTIFICATION FOOD TESTING PERCENT GMO FOOD ANIMAL AND PLANT BREEDING GENE COPY NUMBER
REAL-TIME PCR IN GENE EXPRESSION ANALYSIS EXAMPLE: BRCA1 EXPRESSION PROFILING BRCA1 IS A GENE INVOLVED IN TUMOR SUPPRESSION. BRCA1 CONTROLS THE EXPRESSION OF OTHER GENES. IN ORDER TO MONITOR LEVEL OF EXPRESSION OF BRCA1, REAL-TIME PCR IS USED. Tumour tissue can be assayed to see which BRCA1- controlled genes are being transcribed (if any), and how much they are being expressed compared to non-tumour cells from the same individual
REAL-TIME PCR IN DISEASE MANAGEMENT EXAMPLE: HIV TREATMENT DRUG TREATMENT FOR HIV INFECTION OFTEN DEPENDS ON MONITORING THE VIRAL LOAD . REAL-TIME PCR ALLOWS FOR DIRECT MEASUREMENT OF THE AMOUNT OF THE VIRUS RNA IN THE PATIENT. RNA is reverse-transcribed, then RT-PCR is performed on carefully quantified cDNA sample to see amount of HIV RNA relative to some standard genes. This tells us what treatments may be needed.
Example: Determining percentage of GMO food content DETERMINATION OF PERCENT GMO FOOD CONTENT IMPORTANT FOR IMPORT / EXPORT REGULATIONS. REAL-TIME PCR IN FOOD TESTING LABS USE REAL-TIME PCR TO MEASURE AMOUNT OF TRANSGENIC VERSUS WILD- TYPE DNA. Seed wt DNA GMO DNA
