mRNA Quantification
Real-time PCR To accurately quantify starting amounts of nucleic acid during the PCR reaction without the need for post-PCR analyses. A fluorescent reporter is used to monitor the PCR reaction as it occurs; the reporter can be of a nonspecific or specific nature. The fluorescence of the reporter molecule increases as products accumulate with each successive round of amplification.
RT-PCR Reverse transcription + Polymerase chain reaction mRNA quantification
Threshold Cycle (Ct) In the early cycles of amplification, the change in fluorescence of the reporter is usually undetectable, but at some point during amplification, the accumulation of product results in a measurable change in the fluorescence of the reaction mixture. The point at which the fluorescence rises appreciably above background has been called the threshold cycle.
An Example DCt=log2(fold difference) 3.322=log210 1/10 serial dilution Well Ct B3 10.2 B4 13.6 B5 16.7 B6 20.9
SYBR Green I dsDNA에 결합되지 않은 dye는 아주 약한 형광을 내지만, 컴퓨터 분석과정에서 이러한 결합되지 않은 dye에 의한 형광값(background)은 충분히 제거할 수 있다. Primer를 넣어서 annealing 한 후, 몇몇 dye 분자들이 dsDNA와 결합한다. Dye가 dsDNA에 결합함으로, excitation 후 극격한 형광이 발산한다. DNA가 증폭 되는 동안, dye 분자가 새로이 합성되는 DNA 가닥에 결합한다. 증가되는 형광값을 real-time으로 볼 수 있다.
M****
일단, Ct값이 좀 커서.. Unreliable 하다 10 ng 50 ng 100 ng Reference 25.25 21.8 21.1 HL-60 23.5 21.4 19.5 CCRF-CEM 33.35 30.65 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175 Ct difference Fold-difference 10 ng 50 ng 100 ng HL-60 -1.75 0.4 -1.6 3.36 1.32 3.03 CCRF-CEM 8.1 8.85 274.37 461.44
이것도, Ct값이 좀 커서.. Unreliable 하다 2nd experiment 이것도, Ct값이 좀 커서.. Unreliable 하다 Ct Ct difference fold difference Reference 23.8 HL-60 22.5 -1.3 2.462289 CCRF-CEM 34.6 10.8 0.000561 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
33.35 2.7 22.7=6.50 30.65 25.2 3.4 23.4=10.56 0.7 20.7=1.62 23.5 21.8 21.1 2.1 22.1=4.29 21.4 1.9 21.9=3.73 19.5
2nd experiment 기울기가.. 좀 크다. - 4 정도
A****
이것도 Ct값이 좀 커서.. Unreliable 하다 30.3 2.8 22.8=6.96 차이가 좀 크긴 하네.. 거의 같게 나와야 하는데 27.5 0.7 20.7=1.62 26.8 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
30.3 1.8 21.8=3.48 0.5 20.5=1.41 28.5 28 27.5 1.65 21.65=3.14 25.85 25.2 0.65 20.65=1.57
농도에 따른 경향은 일정한데, 그 slope이 너무 작다. 이론적으로는 3.322가 나와야 한다. Slope of 2.33 means the … 농도에 따른 경향은 일정한데, 그 slope이 너무 작다. 이론적으로는 3.322가 나와야 한다.
mRNA Reference HL-60 100 4.9246 50 3.1383 3.4822 10 1
C****
30.45 27.0 26.45 4 24=16 0.55 20.55=1.464 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
2nd experiment Gene Symbol HL60 CCRF-CEM M difference Fold-difference 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
100 ng/ml 50 ng/ml 10 ng/ml Reference 25.45 25.05 26.45 HL-60 24.25 24.1 27 Ct difference 1.1 0.95 -0.55 Fold-difference 2.14 1.93 0.68 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
mRNA Reference HL-60 100 2.0000 6.2767 50 2.6390 7.4643 10 1
2nd experiment mRNA conc. log (mRNA) Reference HL-60 CCRF-CEM Ct Std. Dev. 100 2 26.3 0.2828 25.1 50 1.699 26.55 0.6364 25.8 10 1 28.65 0.0707 28.8 32.4
2nd experiment
D****
26.45 0.7 20.7=1.62 25.75 0.9 20.9=1.87 24.85 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
100 ng/ml 50 ng/ml 10 ng/ml Reference 24.65 23.95 25.75 HL-60 23.6 23.5 26.45 Ct difference 1.05 0.45 -0.7 Fold-difference 2.07 1.37 0.616 Gene Symbol HL60 CCRF-CEM M difference Fold-difference M**** 1.535745 -1.68172 3.217465 9.301511 A**** -1.96497 -0.09118 -1.87379 0.272856 C**** 1.115822 -1.17847 2.294288 4.905118 D**** -0.13667 0.060641 -0.19731 0.872175
26.45 2.95 22.95=7.73 25.75 1.8 21.65=3.48 - 0.5 2-0.5=0.62 24.65 23.95 23.6 - 0.1 2-0.1=0.933 23.5
mRNA Reference HL-60 100 2.1435 7.2100 50 3.4822 7.7275 10 1
Discussion Repetitive experiments mRNA concentration dependency t-values, are they still valid?