リアルタイムPCR 再入門 - bio-rad.com

1 PCR .. amplification Z5255 0512A. PCR . PCR .. PCR . PCR . amplification PCR . PCR . PCR .. PCR .. amplification PCR . PCR . DNA . (Threshold) .. amplification .. 108,106,104,102 IL-1 ). Threshold Cycle = Ct amplification . Threshold Cycle .. amplification . SYBR Green I .. amplification SYBR Green I. Denature Annealing Extension amplification /TaqMan Denature Annealing Extension amplification /Molecular Beacon Annealing Extension amplification . PCR . /.. amplification / . Primer3. / . /.. RTPrimerDB. qPCR / .. Beacon Designer .. amplification . RNA . /.. A260/A280= RNA . 18S/28S rRNA .. amplification .. /.. amplification .. amplification . SYBR Green I .. amplification .. PCR = [10 (-1/slope) ] - 1.

2 Amplification .. /. - .. - Ct .. amplification . Ct . 100% .. 100% .. amplification .. A 10 pg 10 pg 10/10 = 1. B 60 pg 15 pg 60/15 = 4. A B 4 . amplification . SYBR Green .. 102-108 102-108 . 4 4 . HK TG . A HK A TG . B HK B TG . HK TG . HK = . TG = . amplification Ct / Ct . DCtsampleA = CtTG_sampleA - CtHK_sampleA = 24 12 = 12. DCtsampleB = CtTG_sampleB - CtHK_sampleB = 15 12 = 3. D(DCt) = DCtsampleB - DCtsampleA = 3 12 = - 9. = 2-D(DCt) = 2-(-9) = 512. A B 512 . amplification Ct . SYBR Green . A HK A TG . B HK B TG . HK TG . HK = . TG = . amplification Ct . DDCt Simple 100% . 1 . Pfaffl .. 1 . Vandesompele .. Complex amplification DDCt . DCt control = Ct GOI_control Ct REF_control DCt sample = Ct GOI_sample C tREF_sample D(DCt) = DCt sample - DCt control =control = 2-D(DCt) =sample (Gene of Interest =GOI.)

3 Reference Gene =REF. amplification Pfaffl . (Ct GOI (control) - Ct GOI ). (sample). EGOI. =. (Ct REF (control) - Ct REF (sample)). EREF. E = 1 + (h/100). h % =control 100% E=2 =sample Ct . (Gene of Interest =GOI. Reference Gene =REF. A new mathematical model for relative quantification in real-time RT-PCR. Michael W. Pfaffl. Nucleic Acids Research, 2001, Vol. 29, No. 9 e45. amplification Vandesompele .. (CtGOI(control)-CtGOI(sample)). E(GOI).. (Rel Quant Ref1 * Rel Quant Ref2 * .. * Rel Quant Ref n)1/n Accurate normalization of real-time quantitative RT- =control PCR data by geometric averaging of multiple internal =sample control genes. Vandesompele, De Preter, Filip Pattyn, Bruce (Gene of Interest =GOI.))

4 Poppe, Nadine Van Roy, Anne De Paepe and Frank Reference Gene =REF. Speleman. Genome Biology 2002, 3 amplification .. /.. - . - Ct .. Ct .. amplification PCR .. PCR PCR . Fast PCR .. amplification PCR .. PCR : .. ( ). amplification PCR ? PCR : .. / .. PCR Fast PCR . amplification PCR . ? 250 bp Fast PCR . : Tm 64 69 . (Tm Santa Lucia nearest-neighbor ). / 98 30 . 92 1 . 70 20 .. * : Tm 2-3bp .. amplification . Fast .. 95 10 98 30 .. 95 15 92 1 . 35 x 60 30 x 35. 70 20 .. 72 30 70 .. 72 10 250 bp . PCR .. : 1 28 34 . = 54 . amplification Fast PCR PCR . PCR . 150 250 bp Tm 63 67 Fast . (F) (S) iCycler . iTaq DNA PCR .. : 95 /3 95 /15 62 /30 72 /30 x 35 72 /10 . Fast : 98 /30 92 /1 70 /20 x 35 . amplification PCR.

5 MiniOpticon PCR PCR . = = PCR = 33 . : 98 / 30 92 /1 70 /5 plate read) x 30 . amplification Fast PCR . PCR . 30 40 .. PCR PCR .. -Fast PCR PDF - Web .. amplificatio