RNA isolation from monolayer cell using Trizol reagent

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RNA isolation from monolayer cell using Trizol reagent 김예지 3 rd Semester of Master’s

RNA isolation from monolayer cell using Trizol reagent 김예지 3 rd Semester of Master’s degree Protein Biochemistry Lab (S 301)

Introduction – Why do we study RNA? DNA Transcription Pre-m. RNA uc l N

Introduction – Why do we study RNA? DNA Transcription Pre-m. RNA uc l N m. RNA ei Splicing peptide Translation Difference of Proteins Ribosome → Difference of Gene expression… → Difference of m. RNA !!!

Introduction: What is the difference between DNA and RNA? Can you spot the difference

Introduction: What is the difference between DNA and RNA? Can you spot the difference between ribose and deoxyribose?

Introduction – What is RT-PCR ? Downstream process after RNA isolation. . . r.

Introduction – What is RT-PCR ? Downstream process after RNA isolation. . . r. RNA : 80∼ 85% t. RNA : 10∼ 15% m. RNA`: 1∼ 5% (heterogenous in size, poly(A)tail) 1) RNA 분리 2) c. DNA 합성: reverse transcription 3) PCR

Introduction –RNA analysis/different methods of RNA isolation 1. Northern hybridization - Size and amount

Introduction –RNA analysis/different methods of RNA isolation 1. Northern hybridization - Size and amount of RNA 2. RT-PCR - Amount of RNA , c. DNA synthesis Different ways of isolating RNA from monolayer of cells… 1. Organic extraction method 2. Spin basket format (Kit) 3. RNase protection assay 3. Magnetic particle method - Amount of RNA and mutation detection 4. Direct lysis method

Introduction – principle of RNA isolation 1. Controlling RNase activity - RNase inhibitors -

Introduction – principle of RNA isolation 1. Controlling RNase activity - RNase inhibitors - RNase inactivation Outline of RNA isolation 1. Cell Harvest 2. Addition of Trizol 2. Remove protein - Phenol/Chloroform 3. Separate RNA from DNA - Removal of DNA - Selection of poly(A) RNA 3. Addition of Chloroform 4. Incubate at RT/Centrifuge 5. Addition of isopropyl alcohol/Incubate at RT 6. Centrifugation 7. Wash the RNA pallet with 75% ethanol 8. dry/dissolve RNA with DEPC-treated water

Materials - Cells - Huvec(Human umbilical vein endothelial cells) - HEK 293 T cells(Human

Materials - Cells - Huvec(Human umbilical vein endothelial cells) - HEK 293 T cells(Human embryonic kidney 293 transformed cells) - Trizol - Scraper - Chloroform(for RNA) - Isopropyl alcohol - RNase - free 75% Et. OH - RNase - free DW(DEPC - DW)

3분간 실온에서 incubate 해준다. Sample을 14, 000 rpm에서 15분 간 원심분리 한다. Aquose phase

3분간 실온에서 incubate 해준다. Sample을 14, 000 rpm에서 15분 간 원심분리 한다. Aquose phase : RNA Interphase : DNA Organic phase (phenol/chloroform): Proteins, lipids