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

2. 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

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

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

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

6. 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

7. 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 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 × ml
= 75 ug

8. 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 in 10 mM Tris, pH8.5
4. Pure RNA has an A260/A280 ratio of 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

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

10. 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 ul
10mM dNTP 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) ul
0.1M DTT ul
RNasin ul
RTase ul
Add prepared mixture, mix by vortexing and spin-down

11. 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

12. 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

13. 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

14. Hand writing please…… no copy 제출 기한 월요일 반 : 12월 10일 월요일, 12일 수요일 반환
수요일 반 : 12월 11일 화요일, 13일 목요일 반환
목요일 반 : 12월 12일 수요일, 14일 금요일 반환
레포트 찾아가지 않으면 태도점수 -2점
강지선 (김영준 교수님)
첨단관 201B호
내선)