リアルタイム RT-PCR - takara-bio.co.jp

1 PCR Page 1 of 11 RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RNA cDNA cDNA PCR RT-PCR dT cDNA PCR dT cDNA PCR cDNA RT-PCR PCR PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR PCR RT-PCR RT-PCR total RNA PCR Page 2 of 11 RT-PCR RT-PCR RT-PCR RT-PCR dT 3

2 MRNA PCR dT polyA tail dT PCR polyA tail polyA tail kb PCR polyA tail PCR mRNA PCR Reverse Primer RT PCR dT mRNA mRNA mRNA PCR Page 3 of 11 RT-PCR total RNA mRNA RT-PCR total RNA total RNA mRNA RNA total RNA RNA mRNA total RNA DNA total RNA DNA DNA PCR DNA DNase I DNA DNA DNA

3 DNA DNA PCR mRNA DNase I dT dT cDNA cDNA polyA tail mRNA cDNA RNA total RNA RNA small RNA dT PCR Page 4 of 11 RT-PCR DNase I DNA total RNA DNase I RNase-free DNA DNase I DNase I DNA PCR DNA DNA mRNA PCR DNase I DNA DNA DNA PCR PCR A TB Green PCR PCR

4 DNA Tm GC DNA Perfect Real Time DNA 3' mRNA cDNA PCR Page 5 of 11 RT-PCR 80 150 bp PCR 100 80 150 bp 17 25 17 25 GC 40 60 GC 40 60 GC AT 3' AT DNA GC DNA T/C (polypyrimidine) A/G (polypurine)

5 3' 3' G C 3' GC 3' T 3' 3' G C 3' GC GC 3' T 3 4 G C PCR Page 6 of 11 RT-PCR 3 base 3' 3' Tm Tm Tm Tm 2 C Tm Tm Tm Tm Nearest Neighbor Nearest Neighbor Tm Tm 20 Wallace formula

6 Tm 2 A T 4 G C BLAST DNA NCBI BLAST BLAST DNA PCR Page 7 of 11 RT-PCR RT-PCR RNA DNA DNA PCR RNA DNase I DNA UCSC Genome Browser (Human, Mouse, Rat etc.)

7 Tair Seq Viewer (Arabidopsis) DNA cDNA GC 3' Tm OLIGO Primer Analysis Software (Molecular Biology Insights ) Primer3 PCR Page 8 of 11 RT-PCR Perfect Real Time RT-PCR RefSeq Ensembl Plants TB Green Premix Ex Taq II (Tli RNaseH Plus) RR820A TB Green Fast qPCR Mix RR430A TB Green Premix Ex Taq (Tli RNaseH Plus)

8 RR420A PCR PCR PCR PCR 2 DNA dNTP PCR DNA Mg PCR PCR PCR Hot Start DNA PCR 10 15 Taq PCR PCR PCR ROX BSA 30 40 PCR PCR PCR PCR 6 No template control (NTC)

9 Total RNA cDNA total RNA 1 pg 10 pg 100 pg 1 ng 10 ng 100 ng cDNA 1 PCR PCR P2 PCR 80 120 PCR Page 9 of 11 RT-PCR PCR 35 30 PCR No template control 30 PCR DNA DNA PCR DNA cDNA