RT-PCR analysis 2009.2 생화학 실험 2 조교 : 이 선 민 내선 7699, 첨단과학기술관 201-B 호 신과학원 S438 호.

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RT-PCR analysis 생화학 실험 2 조교 : 이 선 민 내선 7699, 첨단과학기술관 201-B 호 신과학원 S438 호

RT-PCR ? -Reverse transcriptase-polymerase chain reaction -single-stranded RNA is reverse transcribed into cDNA -for the detection and quantification of mRNA

Why we use RT-PCR ? Difference of Proteins… → Difference of Gene expression… → Difference of mRNA !!!

RT primer mRNA cDNA Target RNA target cDNA RNA(rRNA, tRNA 포함 ) cDNA

Genomic DNA contamination -Treat RNase free DNase -Select intron containing primer primer PCR product from mRNA PCR product from genomic DNA

Materials - DEPC-DW - 5X Buffer - 10mM dNTP - 10pM Oligo-dT - 0.1M DTT (reducing agent & Required for RNasin ) - RNasin (RNase inhibitor) - Reverse Transcriptase 200 units/ µl

Protocol O.D.260 = 1 RNA 농도 = 1 X 0.04(μg /μl) X 100(dilution fold) = 4 μg /μl 넣어야 할 RNA 양 = 5μg/4 (μg /μl) = 1.25 μl RNA 1.25 μl + oligo-dT 1ul + DW 8.75 μl =12 μl 260/280 = 1.8~2.0 RNA 정량

Protocol RNase contamination 주의 !!!!!!!!!!!!!!!!!!!!!!!!! 1.Quantitation of RNA by spectrophotometer.  1 OD260 unit equals 40ug/ml 2.RNA 1~2ug  transfer to fresh tube 3.Add oligo-dT 1ul 4.Add DEPC-DW until 11ul (total volume 11ul) 5.65’C for 5min and chill on ice 6.Spin down 7.Add :5x Buffer 4ul 12 ul 0.1M DTT2ul 6 ul RNasin1ul 3 ul dNTP1ul 3 ul 8.Incubate 2 min at 42’C 9.1ul of reverse-transcriptase 3x mix 8 ul mix add to each RNA sample

10. Incubate 50min at 42’C 11. Kill the reaction by heating 15min at 70’C 12. Ready for PCR. (You can keep this cDNA at -20’C) 13. PCR using gene specific primer Protocol

Result GAPDH Gene A Gene BGene C 1. RNA 정량 결과 – OD. 값 RNA 농도 2. RT PCR 결과

1. About quantitation of Nucleic acid…  why does the 1 OD 260 unit different between DNA(50ug/ml) and RNA(40ug/ml)? 2. RNasin is RNase inhibitor. What is the principal of their function? And search for other RNase inhibitors & explain their function(2~3 가지정도 ). 3. What is the quantitative real time RT-PCR? Further study

보고서 제출 직접 써서 작성, 과학원 s438 호 앞 월요일반 :12 월 4 일 금요일 1 시까지 수요일반 :12 월 7 일 월요일 11 시까지 금요일반 :12 월 9 일 수요일 1 시까지 레포트 찾는 곳 : 과학원 s438 호 앞 12 월 11 일 금요일 이후 조교 : 이 선 민 내선 7699, 첨단과학기술관 201-B 호 신과학원 S438 호