はじめてのリアルタイム PCR - takara-bio.co.jp

1 PCR PCR Page 1 of 12 PCR PCR PCR PCR DNA RNA SNP PCR PCR PCR PCR PCR PCR PCR RT-PCR PCR PCR 1 DNA 2 2 DNA PCR PCR PCR PCR Page 2 of 12 DNA PCR DNA Threshold Ct Threshold Cycle Ct Ct PCR PCR Page 3 of 12 PCR PCR PCR PCR PCR PCR PCR DNA PCR PCR 96 Thermal Cycler Dice Real Time System PCR Smart Cycler System Thermal Cycler Dice Real Time System III Applied Biosystems 7500 PCR (Thermo Fisher Scientific ) LightCycler Smart Cycler (Roche Diagnostics ) (Cephied )

2 PCR PCR Page 4 of 12 PCR PCR 2 DNA DNA PCR DNA PCR PCR Page 5 of 12 5 - 5 3 DNA Taq DNA 5 3 PCR PCR Page 6 of 12 CycleavePCR RNA DNA RNase H RNaseH RNA RNA RNaseH SNPs PCR PCR PCR DNA Tm PCR DNA PCR PCR Page 7 of 12 Perfect Real Time RT-PCR RefSeq Ensembl Plants

3 PCR TB Green RT-PCR PCR Thermal Cycler Dice Real Time System II TP900 PCR RT-PCR PrimeScript RT reagent Kit (Perfect Real Time) R037A TB Green Premix Ex Taq II (Tli RNaseH Plus) RR820A total RNA PrimeScript RT reagent Kit (Perfect Real Time) 1. RNA RNA PCR PCR Page 8 of 12 1 ( ) 5 PrimeScript Buffer (for Real Time) 2 l 1 PrimeScript RT Enzyme Mix I l mM Oligo dT Primer 50 M *1 l 25 pmol Random 6 mers (100 M) *1 l 50 pmol total RNA RNase Free dH2O Total 10 l *2 *1 Random 6 mers Oligo dT Primer mRNA cDNA Gene Specific Primer Oligo dT Primer (50 M) l 25 pmol Random 6 mers 100 M l 50 pmol Gene Specific Primer (2 M)

4 L 1 pmol *2 10 l 500 ng total RNA 2. 37 C 15 *3 85 C 5 4 C *3 Gene Specific Primer 42 C 15 PCR 50 C PCR TB Green Premix Ex Taq II (Tli RNaseH Plus) Thermal Cycler Dice Real Time System 1. PCR 1 PCR PCR Page 9 of 12 TB Green Premix Ex Taq II (Tli RNaseH Plus) 2 l 1 PCR Forward Primer 10 M) 1 l M *1 PCR Reverse Primer 10 M 1 l M *1 Template 100 ng 2 l *2 l Total 25 l*3 *1 primer M M *2 template DNA template 100 ng RT-PCR cDNA (RT ) template PCR 10 *3 25 l 2.

5 PCR PCR PCR PCR PCR Tm PCR 3 PCR 95 C 30 PCR 40 95 C 5 60 C 30 PCR PCR RNA PCR PCR Page 10 of 12 PCR PCR 10 PCR mRNA RNA 3 RNA X mRNA X H RNA A B C 3 PCR X H 6 RNA A B C Ct PCR PCR Page 11 of 12 X H Ct *1 Ct *1 1 1 - 1 - 2 10 - 10 - 3 100 - 100 - 4 1000 - 1000 - 5 10000 - 10000 - 6 100000 - 100000 - RNA A - - RNA B - - RNA C - - X H X *1 *1 *2 *3 RNA A RNA B RNA C *1 Ct X *2 RNA X H *3 "1" RNA A RNA A PCR PCR Page 12 of 12 X

6 A "1" B C " " " " PCR PCR PCR