1. RNA isolation from monolayer cell Vascular Genomics Laboratory
using Trizol reagent
Vascular Genomics Laboratory
이 성 운

2. Introduction – Why do we study RNA?
Nuclei
DNA
Pre-mRNA
Transcription
Splicing
mRNA
Ribosome
peptide
Translation
Difference of Proteins
→ Difference of Gene expression…
→ Difference of mRNA !!!

3. Introduction – What is RT-PCR ?
rRNA : 80∼85%
tRNA : 10∼15%
mRNA`: 1∼5%
(heterogenous in size, poly(A)tail)
1) RNA 분리
2) cDNA 합성: reverse transcription
3) PCR

4. Introduction – analysis of RNA
1. Northern hybridization
- Size and amount of RNA
2. RT-PCR
- Amount of RNA , cDNA synthesis
3. RNase protection assay
- Amount of RNA and mutation detection

5. Introduction – principle of RNA isolation
1. Controlling RNase activity
- RNase inhibitors
- RNase inactivation
2. Remove protein
- Phenol/Chloroform
3. Separate RNA from DNA
- Removal of DNA
- Selection of poly(A) RNA

6. Materials - Cells - Huvec(Human umbilical vein endothelial cells)
- HEK 293 T cells(Human embryonic kidney 293 transformed cells)
- Trizol
- Scrapper
- Chloroform(for RNA)
- Isopropyl alcohol
- RNase - free75% EtOH
- RNase - free DW(DEPC - DW)

7. Procedure
실험중 RNase에 의한 RNA degradation을 막기위해 latex glove를 반드시 착용하며 말을 자제합니다.
1. Discard the medium and add 1ml of Trizol (per 5-10 x 106 cells)
2. Scrape the cells with scrapper and pass the cell lysate several times through a pipette
3. Transfer the sample to 1.5ml tube and incubate at RT for 5 min
4. Add 0.2ml chloroform and shake tube vigorously by hand for 15sec
5. Incubate at RT for 3 min
6. Centrifuge the sample at 14,000 rpm for 15min.
7. Transfer the aqueous phase to a fresh tube
8. Precipitate RNA by adding 0.5ml isopropyl alcohol and incubate samples at RT for 10min
9. Centrifuge at 14,000 rpm for 10min
10. Remove the supernatant and wash the RNA pellet once with 1ml of 75% cold EtOH
11. Centrifuge the sample at 14,000 rpm for 10min.
12. At the end of the procedure, briefly dry the RNA pellet
13. Dissolve RNA in RNase-free water and incubate for 10 min at 55 to 60ºC

8. Transfer the sample to a 1.5ml tube and incubate at RT for 5 min
Scrape the cells with a scrapper

9. Add 0.2ml chloroform
Shake the tube vigorously

10. Centrifuge the sample at 14,000 rpm for 15min.
Incubate at RT for 3 min.
Aquose phase : RNA
Interphase : DNA
Organic phase (phenol/chloroform): Proteins,lipids

11. Transfer the aqueous phase
to a fresh tube
Add 0.5ml isopropyl alcohol

12. Centrifuge at 14,000 rpm for 10min.
Incubate the samples at RT for 10min.
Centrifuge at 14,000 rpm for 10min.

13. Wash the RNA pellet with 1ml of 75% cold EtOH
Centrifuge at 14,000 rpm for 10min.

14. Further study – discussion에 쓰세요!!
Results
전기영동 결과를 보고 분리해낸 각각의 RNA band가 무엇을 나타내는지 추측해보시오.
Further study – discussion에 쓰세요!!
1. DEPC-DW는 무엇이며, 그것을 사용하는 이유는 무엇인가?
2. Trizol 을 이루는 구성물질과 그것의 기능은 무엇인가?
3. Huvec과 293T의 RNA 양은 왜 다를까?
4. Isopropyl alcohol은 상온에서 75% Ethanol은 cold상태로 쓰는 이유는 무엇이며 alcohol precipitation을 하는 이유를 쓰시오.
5. Monophasic phenol과 chloroform을 5:1 로 넣어주는 이유는?
6. RNA -> DNA -> PROTEINS,LIPIDS 로 나뉘는 이유는?
조교 : 이 성운
010 – 8714 – 1983