• Document: Quantitation of mrna Using Real-Time Reverse Transcription PCR (RT-PCR)
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Quantitation of mRNA Using R l Ti Real-Time Reverse R Transcription p PCR (RT-PCR) ( ) Quantitative Real-Time RT-PCR V Versus RT RT-PCR PCR • In Real-Time Real Time RT- RT PCR, PCR DNA amplification monitored at each cycle but RT-PCR measures the amount of accumulated PCR product at the end of the program, • Real-Time PCR is quantitative, traditional PCR is qualitative. Real-Time PCR Instrumentation Growing Use of Real-Time PCR f the for h Years Y off 1990–2004 1990 2004 http://advan.physiology.org/content/29/3/151/F1.expansion.html PCR Steps (Overview) • Sample (tissue/cell) Preparation, • RNA/DNA Extraction/Purification, • cDNA DNA synthesis, th i • RT-PCR , p , • Electrophoresis, • Documentation, • Data Analysis. PCR (Overview) primers extend polymerase anneal denat. extend anneal denat. DNA etc… 95oC 55-60oC 72oC 95oC 55-60oC 72oC Adopted from: http://www.dnalc.org/ddnalc/resources/pcr.html What are the reagents in Real- Time PCR tubes • DEPC (Diethylpyrocarbonate) water, • Master Mix (Buffer, dNTP, Mgcl2),???? • Primers (Forward, Reverse), • Amplicon (target sequence, sample), • Termocycler (Real-Time PCR machine). Point: buffers should stay out of light light- Why??? . Master Mix (qPCR Product Detection) • There are many fluorescence detection strategies to monitor PCR product accumulation during PCR • Th Those which hi h use a d dye e.g. SYBR G Green, Eva Green, Disadvantages: not specific – detects all double-stranded products. Advantages: i inexpensive, i convenient, i t just j t as sensitive iti as a fluorescent fl t probe. Master Mix (qPCR Product Detection) • Those which use a sequence specific probe which is exactly complementary to the PCR product sequence. Many probe formats available ( Scorpion, p FRET, M. beacon). ) Disadvantages: Expensive, need new probe for every gene measured, cannot do melting analysis to confirm PCR product identity. Advantages: Sequence q specific p detection,, p potential for multiplex p q qPCR. By far the most common methods are SYBR Green detection, and hydrolysis probes assays ( (e.g. T M ) TaqMan). Detection strategies g – SYBR Green SYBR Green Exciting g light g Fluorescent emission signal 3' Fd Fd 3' 5' Target strand kate kirwan 2003 Detection strategies – SYBR Green http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/real Real-Time PCR Assay y Design g • Incubate at 95C, 5 min, • Incubate at 95C ,1 min, • Incubate at optimized annealing temp (for each primer set) 15S - 1min, • Plate Read, Read • Goto line 2 for 40 more times, • Melting Curve from 50C to 95 C, read, every 1.0 C, hold 10S • END. END Quantitative Real-Time PCR Amplification Plot What does these curves mean?? Cycle Threshold (CT Value) Threshold Lines Point: Placement of threshold line affects Cq (CT Value) Cycle y Threshold ((CT Value))  Hi High h Concentration C t ti  Medium Concentration  Low Concentration Point: RT-PCR is semi-quantitative SYBR Green Melt Analysis F Raw data plot: Fluorescence (F) d

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