2018 2 Reverse transcription PCR RealTime PCR Cell

2018 -2학기 생명과학실험기법 Reverse transcription PCR & Real-Time PCR

Cell culture Seeding Treatment RNA extraction c. DNA synthesis Real-time PCR

1. RNA extraction cell 2. Concentration measurement

3. c. DNA synthesis 1. 역전사효소 (reverse transcriptase) 2. 5 X reaction buffer (d. NTPs, Mg 2+ …) 3. RNA 4. Nuclease free water(to 20μl) c. DNA 합성 Kit

4. real-time PCR의 원리 Real-time PCR의 증폭곡선

Real Time PCR Method (SYBR green vs Taq. Man) Taq. Man SYBR Green • • SYBR Green 1 fluoresces only when bound to ds. DNA Taq. Man requires a sequence-specific probe that connects fluorophore and quencher specificity < expense < Quencher DNA polymerase의 exonuclease 기능에 의해 Taq. Man probe 로부터 flurophore 가 분리되어 발광함

c. DNA synthesis Method 1. E-tube에 mixture를 만든다. ea Total 4 ul 16 i. Script reverse transcriptase 1 ul 4 DW 6 ul 24 5 X i. Script reaction 2. Pcr tube에 RNA, mixture, and DW를 넣어준다. AML-12 ng/ul Average 1000 ng->? ul DW Total Control 9 Compound-1 9 Compound -2 9 3. PCR machine 으로 c. DNA합성 1) Priming 5 min at 25’C 2) Reverse Transcription 20 min at 46’C 3) RT inactivation 1 min at 95’C 4) Optional step Hold at 4’C 3. c. DNA 1/10 dilution : RNA 1μg로부터 동량의 c. DNA가 합성되었다고 가정하면, 합성된 총 c. DNA는 1μg/20μL = 50 ng/μL 이후 real-time PCR에 사용되는 c. DNA template는 20 ng 사용함. (합성된 c. DNA 20 u. L + 180 u. L nuclease-free water = 200 u. L)

Method Real-time PCR 1. 96 well PCR plate의 정해진 위치에 c. DNA 4 ul 씩 loading DNA 4 ul 2 XSYBR 10 ul 40 Primer (Forward) 0. 2 ul 0. 8 Primer(revers e) 0. 2 ul 0. 8 DW 5. 6 ul 22. 4 Total(ul) 20 80 2. Mixture (primer, DDW, SYBR Green) 16 ul loading Primer(Target gene) Primer(Reference Gene ) Primer : β-actin Primer : PPARα 3. Centrifuge 1000 rpm, 2 min 4. Real time PCR machine 에 넣고 작동시킨다.

Result Ex) Gene X 9 Relative m. RNA level 8 7 6 5 4 3 2 1 0 A B