RT-PCR analysis Genome Regulation Center 최원영 ( 김영준 교수님 실험실 ) 첨단관 201B 호 H.P :

Reverse Transcriptase RNA-dependent DNA polymerase (RdDP) DNA polymerase that transcribes ssRNA into dsDNA Retrovirus (i.e. HIV, MLV)

RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction) - single-stranded RNA is reverse transcribed into cDNA - for the detection and quantification of mRNA

mRNA → cDNA target RNA → target cDNA Whole RNA → cDNA RT primer design

RNA quantitation Determination of nucleic acids using spectrophotometry Purines(A, G) and pyrimidines(T, C) in nucleic acids absorb UV light (260 nm). A260 (OD260) of 1 corresponds to -50 ug/ml for dsDNA -40 ug/ml for ssRNA -33 ug/ml for ssDNA ※ Interference: EDTA, Phenol, Urea and Organic compounds

Determination of Nucleic Acid Concentration 1. Measure DNA or RNA concentration using the A 260 value. Please note that this measurement does not discriminate between RNA and DNA. 2. Water is recommended as the solvent for measuring DNA or RNA concentration 3. Use the same solvent to zero the spectrophotometer before measuring the sample 4. Ensure that the cuvettes are Rnase-free for measuring RNA samples. Example: Measuring RNA concentration Volume of RNA = 50 ul Dilution: 10 ul RNA sample ul distilled H 2 O (1/50 dilution) A 260 of diluted sample (1 cm length) = 0.75 RNA concentration = 40 ug/ml × A 260 × dilution factor = 40 ug/ml × 0.75 × 50 = 1500 ug/ml Total amount of RNA = concentration × volume of sample in ml = 1500 ug/ml × ml = 75 ug

{ Determination of Nucleic Acid Purity 1. Measure the nucleic acid purity using A 260 /A 280 ratio 2. Use low salt buffer as they provide a more accurate measurement. Purity is influenced by pH; lower pH solutions lower the A 260 /A 280 ratio and reduce the sensitivity to protein contamination 3. Pure DNA has an A 260 /A 280 ratio of Pure RNA has an A 260 /A 280 ratio of Detecting Contamination 1. Absorbance at 230 nm and 270 nm indicates the presence of phenol, urea, thiocyanates and other organic compounds 2. Absorbance at 280 nm (hence, a low A 260 /A 280 ratio) indicates the presence of protein 3. Absorbance at 325 nm indicates contamination by particles and/or dirty cuvettes

Nano drop

DEPC-DW 5X FSB (First Strand Buffer) 10mM dNTP 10pM Oligo-dT 0.1M DTT (Reducing agent) RNasin (RNase inhibitor) Reverse Transcriptase 200 units/ µl Materials

Protocol 모든 procedure 는 ice 에서 !!! RNase contamination 주의 !!!!!!!!!!!! 1.Add the following components to a nuclease-free e-tube: 2ug of total RNA x λ 10pM oligo dT 1 λ 10mM dNTP 1 λ DEPC-DW (10-x) λ 2. Heat mixture to 65 ℃ for 5min and quick chill on ice. During the heating time prepare following mixture: 5X FSB (first-strand buffer) 4 λ 0.1M DTT 2 λ Rnasin (Rnase inhibitor) 1 λ 3.Add prepared mixture and incubate at 42 ℃ for 2min 4.Add 1ul of RTase, mix by vortexing and spin-down 1 reaction

5.Incubate at 42 ℃ for 2hrs 6.Inactivate the reaction by heating at 75 ℃ for 15min ⇒ Ready for PCR. (You can keep this cDNA at -20 ℃ ) 7. PCR using gene specific primer Usual Reverse transcription program RT 1RT 2

Result RNA 정량 결과 : 농도, isolation 된 total RNA 양, purity (260/280) RT-PCR 결과 : 올려준 gel 사진 첨부 Discussion 실험 과정에 대한 설명 (step 마다 각 reagent 를 넣어준 이유, 왜 그 온도에서 incubation 하는가 ?) RT-PCR 결과에 대한 해석

Further study 1. OD 260 의 값이 1 일 때, DNA(50ug/ml) 와 RNA(40ug/ml) 의 extinction coefficient 값의 차이가 나는 이유를 간단히 서술하시오. 2. Find another control gene to compare gene expression pattern between samples (2-3 gene is enough-except GAPDH, Briefly explain their function) -Introduction 과 Method 는 핵심적인 것만 정리해서 적어주세요. - 실험 결과 자체는 채점대상이 아닙니다. 하지만 결과가 없다면 ㄱ Result 에 대한 해 석과 그에 대한 충분한 discussion 을 적어주시기 바랍니다.