实时荧光定量PCR原理与分析方法 Real-time, fluorescence …

1 Good Science Good Products PCR Real-time, fluorescence -based quantitative PCR /Good Products ,Good Science 1 qPCR 2 3 qPCR 4 qPCR /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science SNP Main advantage:the starting DNA concentration in a closed-tube systemqPCR /Good Products ,Good Science DNA intercalatingmoleculesfluorescent reporter moleculesfluorophore-labeled oligonucleotides binds to the minor groove of dsDNA non-specific extension phase of each cycle of qPCR hydrolysis probe specific PCR products extension phase qPCR chemistry /Good Products ,Good Science Clin Chim Acta.

2 2015 Jan 15;439:231-50. DNA intercalating moleculesfluorophore-labeled oligonucleotides (Taqman)StructureIntercalating dye5 R---Q3 Advantages Lower costs melting curve analysis Specific more sensitive and reproducible(Lowly-expressed gene) Disadvantages non-specific Expensive primer-dimers might be formed Applicationspathogen detection, gene expression, mutation detection, SNP detection virus detection , viral/bacterial load quantitation, genotyping, allelic discrimination, mutation detection, SNP detection, gene expression analysis SYBR Green I VS TaqMan Probe /Good Products ,Good Science Determining Starting template concentration qPCR /Good Products ,Good Science qPCR VS PCR: qualitative or quantitative A BA B A=3BA B A B qPCR PCRA B PCR showing the presence or absence of the DNA sequence of interest quantitativeqPCR /Good Products ,Good Science Determining Starting template concentration qPCR /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science n 1/ 2/ 3/ qPCR n 1/ Seven Key StepsqPCR /Good Products ,Good Science Ct Value and Ct 3 /Good Products ,Good Science n nCT Cycle2.

3 NNTC NRC n nNTC NRC n n 90-110%nR2nNTC NRC qPCR Ct value and Ct /Good Products ,Good Science Ct CT Ct Value /Good Products ,Good Science Rox Thermo /Good Products ,Good Science /Good Products ,Good Science Thermo Target /Good Products ,Good Science Thermo /Good Products ,Good Science /Good Products ,Good Science /Good Products ,Good Science /Good Products ,Good Science /Good Products ,Good Science Ct /Good Products ,Good Science TM Tm Ct Value /Good Products ,Good Science Xn = X0 2n ; Xn = X0 (1+E )n ; Xn Log X0 = LogM-CTLog (1+E )Determining Starting template concentration R2 90%-110%Ct Value /Good Products ,Good Science 1.

4 2. 3. qPCR 4. y= + = %= 10-(1/ -1 100%= Log amount of standardCtInitial concentration of unknownR2 90%-110% /Good Products ,Good Science gene expression analysis: Ct 100% GAPDH Actin 18S rRNA Ct /Good Products ,Good Science Ct /Good Products ,Good Science qPCR n 1/ 2/ 3/ qPCR /Good Products ,Good Science qPCR RIN RNA integrity number RIN > 7 6-7 < 6 Agilent 2100 Bioanalyzer RNA /Good Products ,Good Science Sample Acquisition & Handling: Avoid cell contamination Rapid sample handling Avoid RNase contaminationRNA Extraction: Avoid RNase contaminationRNA Assessment: RNA purity: A260/280 ~ ; A260/230 ~ RNA integrity: RIN agarose gel DNA RNA /Good Products ,Good Science Reverse Transcription Protocol RNA gDNA RNAcDNAT otal RNA: 100ng-1 g20 l 20 l 5 -10 qPCR1-2 l qPCR /Good Products ,Good Science 1A.)

5 CDNA 2. RNA cDNA 1B. cDNA /Good Products ,Good Science Primer designAmplification efficiencycDNA concentration90% 110%NTC/NRC no template control:only water. no RTase control qPCR qPCR Optimisationthe expression level of the target value (20 to 30) in triplicate /Good Products ,Good Science Primer design and testingdetermines amplicon length, melting temperature and amplification efficiency Specificity, Length, GC content, 3 end stability, sequence complexity, melting temperature, and location in the target sequence of the primers Primer3 Primer-BLAST secondary structure of primersmFold /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science 1 /Good Products ,Good Science 2 Ct 1.

6 2. 100-300bp 4. /Good Products ,Good Science 3 CT 1. 2. 3. 4. STD /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science PCR 123 4 /Good Products ,Good Science Calculation of TNF expression levels in Jurkat cells using the CT method Jurkat cells were untreated or treated with : TNF in Jurkat cells is very low level. Qiagen /Good Products ,Good Science Calculation of TNF expression levels in Jurkat cells using the CT method Jurkat cells were untreated or treated with : TNF in Jurkat cells is very low level. Qiagen 1 Ct 1.

7 2. 2 CT 1. /Good Products ,Good Science 1 . 100% 2 . /Good Products ,Good Science /Good Products ,Good Science First Second: Ct Third Fourth: /Good Products ,Good Science Microwell-Seq 50 40 qPCR HieffTM qPCR Master Mix1. 3. 2. Good Science Good Products Qusition!