リアルタイムRT-PCR実験法 - takara-bio.co.jp

1 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 1 of 11 RT-PCR . RT-PCR .. RT-PCR .. RT-PCR dT . 3 .. mRNA PCR.. dT . poly(A) tail dT . PCR poly(A) tail . poly(A) tail kb PCR. poyl(A) tail PCR . mRNA .. PCR Reverse Primer .. RT-PCR dT . mRNA mRNA . mRNA . PCR Page 2 of 11 RT-PCR .. dT . dT cDNA cDNA poly(A) tail mRNA cDNA .. RNA . total RNA RNA small RNA . dT .. 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 DNA .. DNA . PCR mRNA .. DNase I . PCR Page 3 of 11 RT-PCR . DNase I DNA . total RNA DNase I RNase-free DNA DNase I DNase I.

2 DNA . PCR DNA . DNA mRNA PCR . DNase I DNA . DNA . DNA PCR.. DNA 3' . mRNA cDNA .. PCR. A SYBR Green .. PCR PCR .. DNA . Tm GC .. DNA .. Perfect Real Time .. PCR Page 4 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) .. 3' . 3' G C . 3' GC . 3' T . 3' 3' . G C 3' GC . GC . 3' T .. 3 4 G C .. PCR Page 5 of 11 RT-PCR .. 3 base . 3' .. 3' .. Tm . Tm . Tm . Tm 2 . Tm Tm . Tm .. Tm Nearest Neighbor . Nearest Neighbor Tm . Tm 20 Wallace formula .. Tm 2 A T 4 G C .. BLAST . DNA . NCBI BLAST . BLAST . DNA .. PCR Page 6 of 11 RT-PCR . RT-PCR .. RNA DNA DNA PCR . RNA . DNase I DNA .. UCSC Genome Browser (Human, Mouse, Rat etc.). tair Seq Viewer (Arabidopsis).. DNA . cDNA .. GC 3' . Tm .. OLIGO Primer Analysis Software ( NB100 NB100W).

3 Primer3 . PCR Page 7 of 11 RT-PCR . Perfect Real Time . RT-PCR . RefSeq . SYBR Premix Ex Taq (Perfect Real Time) ( RR081) SYBR Premix Ex Taq (Perfect Real Time) ( . RR041) PCR . PCR . RefSeq . Affymetrix GeneChip . 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) . total RNA cDNA . total RNA 1 pg 10 pg 100 pg 1 ng 10 ng 100 ng . cDNA 1 . PCR . PCR Page 8 of 11 RT-PCR . PCR P2 PCR 80 120 .. PCR . SYBR Green I . 35 . 30 PCR.. No template control 30 . PCR . DNA . DNA PCR DNA . cDNA .. total RNA DNase I RTase .. PCR .. PCR . PCR .. B TaqMan . TaqMan . TaqMan .. TaqMan . TaqMan . SYBR Green TaqMan SYBR Green I . TaqMan .. TaqMan . PCR Page 9 of 11 RT-PCR .. RNase H . RNA/DNA RNA RNase H RNase H.

4 CDNA RT-PCR . RT-PCR RNase H . RNase H . RNase H PCR . BioTechniques 32: 1224 1225 (2002) .. RT-PCR PrimeScript RT reagent Kit (Perfect Real Time) Random primers Oligo dT primer . cDNA cDNA/RNA . PCR . 30 1 PCR. DNA .. UNG . UNG PCR . PCR PCR 100 bp . PCR PCR . UNG .. UNG (Uracil-N-Glycosylase) . UNG DNA DNA U PCR . dTTP dUTP PCR U . PCR PCR PCR UNG .. PCR Page 10 of 11 RT-PCR .. PCR RT-PCR . RNA . RNA . PCR .. N .. PCR Page 11 of 11 RT-PCR.