文件類別
作業流程
Protocol ID
Protocol Name
Total RNA Isolation
Material：
Commercial Kit/ Reagent
Item
ID
Name
1
TRIzol Reagent
2
Chloroform
3
Isopropyl alcohol
4
Glycogen
Part No
Brand
Specification
Stock conc.
Storage
Working conc. X amount
Reagent
Item
ID
Reagent Name
1
Ethanol (RNase-free)
2
DEPC-treate Water (RNase-free)
3
PBS solution
4
Trypsin
95%
75%
1x,
Plastic ware
Item
ID
Name
1
Centrifuge tube
2
Eppendrof tube
Part No
Brand
Specification
Cat.430766
Corning
15 ml
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1.5 ml
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文件類別
作業流程
Protocol ID
Protocol Name
Total RNA Isolation
Method：
Sample Homogenization:
1. Tissues:
a. Cut the tissue into pieces.
b. Grind the chopped tissues into powder using mortar in the liquid nitrogen-chilled condition.
c. Add TRIzol (1 ml per 50-100 mg of tissues) to the 15 ml-centrifuge tube (Corning), then transfer the
powdered tissues to the tube.
2. Cultured adhered or monolayer cells:
a. Wash cells twice with 1x PBS.
b. Suspend cells by trypsin treatment.
c. Collect the suspended cells in 1x PBS, and transfer to a centrifuge tube.
d. Spin down cells at 1,000 rpm for 5 min.
e. Disperse the pellet by flicking the tube.
f. Add TRIzol to the cell pellet (1 ml of TRIzol per 5-10x106 cells), pipette the resulting cell lysate several
times gently.
Note: If you are using culture dishes smaller than 3.5cm in diameter, step b can be skipped.
Simply add
TRIzol to the washed cells directly (1ml TRIzol per 10cm2).
3. Cells Grown in Suspension:
a. Pellet cells by centrifugation.
b. Disperse the pellet by flicking the tube.
c. Add TRIzol to the cell pellet (1 ml of TRIzol Reagent per 5-10x106 of cells) and repeat pipetting.
Total RNA isolation:
1. Incubate the homogenized sample for 5-10 min at room temperature. (If you are not using the sample
immediately, freeze the homogenized materials at -60 to -70℃ until use.)
2. Add 0.2 ml chloroform to the TRIzol-treated cell lysate (per 1 ml), and shake vigorously by hand. Incubate the
mixed solution at room temperature for 5-10 min.
3. Centrifuge the tube at  12,000 rpm for 15 min at 4℃.
4. Obtain the upper phase of the solution carefully (about 0.6 ml), and transfer the solution to a fresh 1.5 ml
eppendrof tube.
5. Add 0.5 ml isopropyl alcohol to the tube (per 1 ml initial TRIzol cell lysate).
Mix thoroughly and incubate at
room temperature for 10 min. (For samples less than 50 mg or 5x106 cells, add 0.5 ml isopropyl alcohol and
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文件類別
作業流程
Protocol ID
Protocol Name
Total RNA Isolation
2.5-5g glycogen to the sample, incubate at -20℃ for 30 min, then go to step 6)
6. Centrifuge the tube at  12,000 rpm for 10 min at 4℃.
7. Discard the supernatant. Wash the pellet (containing total RNA) twice with 1 ml 75% pre-chilled (-20℃)
ethanol.
8. Remove the supernatant ethanol completely.
9. Spin down the tube and remove the ethanol adhered to the tube wall.
10. Dry the RNA pellet (air-dry or vacuum-dry) for 5-10 min. Prevent over drying the RNA pellet (i.e., the
“while-colored” RNA pellet should contain a transparent rim).
11. Re-suspend the RNA in RNase-free water (DEPC water).
12. Measure the 260/280 ratio (indicating protein contamination) and quantify the RNA concentration.
Note：
1.
Wear disposable gloves at all times.
2.
Use sterile, disposable and/or autoclaved plastic ware and tubes for all RNA works.
3.
Prevent cross-contamination of RNases from sharing equipment.
4.
If going directly from TRIzol isolated total RNA to cDNA synthesis, it may be beneficial to perform a second
cleanup using QIAGEN RNeasy Mini Kit.
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