Real-time RT-PCR
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA isolation from animal cell cultures - Spin Protocol
( RNeasy Mini Kit, Qiagen, Valencia, CA)
All steps of the RNeasy protocol should be performed at room temp. (20 – 25 ºC).
Before starting,
Add 10
l
-mercaptoethanol (
-ME) per 1 ml Buffer RLT [Buffer RLT is stable for 1 month after addition of
-ME].
Before using for the first time, add 4 volumes of ethanol to Buffer RPE to obtain a working solution.
Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature.
Wash monolayer cells on 100-mm [or 60-mm] culture dish with HBSS or PBS once.
Add 600
l [or 300
l for 60-mm dish] of Buffer RLT to culture dish directly and collect cell lysates with a cell scrapers.
Pass several times by pipetting, transfer the lysates directly onto a QIAshredder spin column (Qiagen) placed in a 2 ml collection tube (supplied), and centrifuge for 2 min at maximum speed ( 14,000 rpm ) to homogenize the sample [
form homogenous lysate].
Add 600
l or [300
l for 60-mm dish] of 70% ethanol to the lysates and mix well by pipetting.
Apply 700
l of the sample to an RNeasy mini column placed in a 2 ml collection tube (supplied).
Close the tube gently, centrifuge for 15 sec at ≥ 8,000 × g (or ≥ 10,000 rpm ), and discard the flow-through [100-mm dish: repeat this step one more time].
Transfer the RNeasy column into a new 2 ml collection tube (supplied) and add 500
l Buffer RPE onto the RNeasy column.
Close the tube gently, centrifuge for 15 sec at ≥ 8,000 × g (or ≥ 10,000 rpm ) to wash the column, and discard the flow-through.
1 Dr. Lee’s Lab
Add another 500
l Buffer RPE to the RNeasy column, close the tube gently, centrifuge for 2 min at ≥ 8,000 × g (or ≥
10,000 rpm ) to dry the RNeasy silica-gel membrane, and discard the flow-through.
Place the column in a new 2 ml collection tube, and centrifuge for 1 min at full speed
( 14,000 rpm ) to eliminate any chance of possible Buffer RPE carryover.
To elute, transfer the column to a new 1.5 ml collection tube (supplied), add 30 ~ 40
l RNase-free water directly onto the silica-gel membrane, and centrifuge for 1 min at ≥ 8,000 × g (or ≥
10,000 rpm ) to elute.
To obtain a higher total RNA concentration, use the first eluate for the second elution step, and centrifuge for 1 min at ≥ 8,000 × g (or ≥ 10,000 rpm ).
Determination of RNA concentration by spectrophotometer.
Adjust 0.5
g total RNA/
l
Reverse Transcription Reaction!
2 Dr. Lee’s Lab
Isolation of total RNA from cell cultures or tissues - TRI REAGENT
1.
Lysis of animal cell cultures or tissues
Wash monolayer cells (on 100 mm culture dish) with HBSS or PBS once.
Add 1 ml of TRI REAGENT (Sigma T9424) to culture dish or tissues directly.
Cell cultures: Passing several times through a pipette.
Tissues: Homogenizing by ultrasonication (most tissues) or Bullet Blender (Aortas).
[
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
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.
Adjust 0.5
g total RNA/
l
Reverse Transcriptase Reaction!
3 Dr. Lee’s Lab
Reverse transcription
[Reverse Transcription System, Promega, Madison, WI]
[ final concentation]
RNase-free water 7.75
l
MgCl
2
4
l [ 5 mM ]
RT buffer 10X 2
l 1 X
dNTP mixture 2
l [ 1 mM each NTP ]
Random hexamers 1
l [ 0.5
g ]
RNasin 0.5
l [ 1 unit/
l ]
AMV RT 0.75
l [ 15 unit/
g ]
Prepare the master mix
Master mix 18
l + total RNA 2
l (1
g) [ total 20
l ]
25
C for 10 min, 42
C for 45 min, 99
C for 5 min, and quickly on ice for 5 min
Real-time PCR
[In case using TaqMan® Gene Expression Assays as target primers and probe]
RNase-free water 9.25
l
20X Target Primers and Probe* 1.25
l
2X PCR Master Mix** 12.5
l
[*TaqMan® Gene Expression Assays, Applied Biosystems, Foster City, CA]
[**TaqMan® Universal PCR Master Mix, Applied Biosystems, Foster City, CA]
Prepare the master mix
Master mix 23
l + cDNA (RT reaction) 2
l [ total 25
l ]
Setting Thermal Cycler Conditions
Initial steps: 50
C for 2 min, 95
C for 10 min
Each of 45 cycles: 95
C for 15 sec, 60
C for 1 min
Analysis by Applied Biosystems 7300 Real-Time PCR System
4 Dr. Lee’s Lab
