Genome Regulation Center RT-PCR analysis Genome Regulation Center Kang Ji-Seon

RT-PCR (Reverse Transcription Polymerase Chain Reaction) single-stranded RNA is reverse transcribed into complementary DNA (cDNA) for the detection and quantification of mRNA (mRNA expression level) DNA qPCR cDNA

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

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

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

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

Determination of Nucleic Acid Concentration 1. Measure DNA or RNA concentration using the A260 value. Please note 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 + 490 ul distilled H2O (1/50 dilution) A260 of diluted sample (1 cm length) = 0.75   RNA concentration = 40 ug/ml × A260 × dilution factor = 40 ug/ml × 0.75 × 50 = 1500 ug/ml Total amount of RNA = concentration × volume of sample in ml = 1500 ug/ml × 0.050 ml = 75 ug

Determination of Nucleic Acid Purity 1. Measure the nucleic acid purity using A260/A280 ratio 2. Use low salt buffer as they provide a more accurate measurement. Purity is influenced by pH; lower pH solutions lower the A260/A280 ratio and reduce the sensitivity to protein contamination 3. Pure DNA has an A260/A280 ratio of 1.8-2.0 in 10 mM Tris, pH8.5 4. Pure RNA has an A260/A280 ratio of 1.9-2.1 in 10 mM Tris, pH8.5 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 A260/A280 ratio) indicates the presence of protein 3. Absorbance at 325 nm indicates contamination by particles and/or dirty cuvettes

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

Protocol 모든 procedure는 ice에서! RNase contamination 주의! Add the following components to a nuclease-free e-tube: 3ug of total RNA x ul 10pmole oligo dT 1 ul 10mM dNTP 1 ul DEPC-DW (10-x) ul 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 ul 0.1M DTT 2 ul RNasin 1 ul RTase 1 ul Add prepared mixture, mix by vortexing and spin-down

4. Incubate at 42℃ for 50min Inactivate the reaction by heating at 70℃ for 15min ⇒ Ready for PCR. (You can keep this cDNA at -20℃) 6. PCR using gene specific primer

Result RNA 정량 결과 : 농도, isolation된 total RNA양, purity (260/280) RT-PCR 결과 : 올려준 gel 사진 첨부 Discussion 이해한 대로 RT-PCR 과정에 대한 그림을 그려서 실험에 대한 설명 (step마다 각 reagent를 넣어준 이유, 왜 그 온도에서 incubation 하는가?) RT-PCR 결과에 대한 해석

Further study 1. Why does the 1 OD260 unit different between DNA(50ug/ml) and RNA(40ug/ml)? 2. Explain about DTT and RNasin. 3. What is the quantitative real time PCR (qRT-PCR, qPCR)? Explain each method with pros & cons. : SYBR-Green, TaqMan probe and Cycling probe

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