Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Isolation of total RNA for RT-PCR using TRI REAGENT
[ Improvement of the single-step method reported by Chomczynski and Sacchi for total
RNA isolation ( guanidine thiocyanate and phenol method ) ]
1. Lysis of cultured monolayer cells
Wash monolayer cells (on 100 mm culture dish) with HANKS once
 Add 1 ml of TRI REAGENT (Sigma T9424) to culture dish directly
 Passing several times through a pipette [ form homogenous lysate ]
 Centrifuge the homogenate at 12,000  g for 10 min at 4 C to remove the insoluble
material
 Transfer the clear supernatant to a microcentrifuge tube and allow samples to stand
for 5 min at room temperature
[* After the cells have been lysed in TRI REAGENT, samples can be stored at –70 C
for up to 1 month]
2. Phase separation
Add 0.2 ml of chloroform and shake vigorously for 15 sec
 Stand for 10 min at room temperature and centrifuge (12,000  g, 15 min, 4 C)
 Transfer the colorless upper aqueous phase (RNA part) to a fresh tube
[interphase ; DNA part, lower layer ; protein part]
3. RNA precipitation
Add 500 l of isopropanol, mix, and stand for 10 min at room temperature
 Centrifuge (12,000  g, 10 min, 4 C) and wash the RNA pellet by adding 1 ml of
75% ethanol [vortex, centrifuge at 12,000  g for 5 min, 4 C]
 Briefly dry the RNA pellet for 10 min by air-drying
[* Samples can be stored in ethanol at 4 C for at least 1 week and up to 1 year at –20 C]
4. RNA solubilization
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Dr. Lee’s Lab
Add 40 l of nuclease-free water to the RNA pellet
 Mix by repeated pipetting with a micropipette at 55 – 60 C for 10 min
 Incubate for 5 min at 70 C and chill quickly on ice
 Determination of RNA concentration by spectrophotometer
[OD260/OD280 ratio  1.7 ]
 Adjust 1 g total RNA/ l  Reverse Transcriptase Reaction !
Reverse transcription (Promega, RT System, A350)
[ final concentation]
DW
8.75 l
MgCl2
4 l
RT buffer 10X
2 l
dNTP mixture
2 l
Oligo(dT)15
1 l
[ 0.5 g ]
RNasin
0.5 l
[ 1 unit/l ]
AMV RT
0.75 l
[ 15 unit/g ]
[ 5 mM ]
[ 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100 ]
[ 1 mM each NTP ]
Prepare the master mix
 Master mix 19 l + total RNA 1 l (1 g) [ total 20 l ]
 42 C for 60 min, 99 C for 5 min [User # rt], on ice for 5 min  “PCR”
Polymerase chain reaction (Qiagen, Taq PCR Master Mix Kit, 201443)
DW
21 l
Sense primer
1 l (20 pmol/50 l)
Antisense primer
1 l (20 pmol/50 l)
RT product
2 l
Taq PCR Master Mix
25 l
 Mix well and PCR !
2% Agarose gel electrophoresis
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Dr. Lee’s Lab
Staining gel with SYBR Green for 1 h
Analysis by Phosphor Imager
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Dr. Lee’s Lab