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Total RNA Extraction
Reagent required:
Clorofoam
Isopropyl Alcohol
75% Ethanol
RNase free water or 5% SDS solution
Safety: Wear Gloves and Lab Coat
Preparations:
1. Use 1M NaOH to clean probe for 15 min., screw back in rinsing with ethanol and water
drying it completely.
2. Use 70% ethanol to clean centrifuge and replace silver rotor with black rotor (located in
RNA Zone).
3. Set centrifuge to 5˚C at 11500 rpm for 15 min. (see step #9)
Procedure:
1. Label microcentrifuge tubes (tubes at RNA Zone).
2. Obtain tissue samples and place into tubes (embryos to 0.1 marks, if older then to 0.5
marks, unsure ASK INSTRUCTOR)
3. Draw out excess Hank’s solution by pipetting 10 μl
4. Add 1000 μl (1ml) TRIzon Reagent
5. Sonicated 15 small short shocks or til dissolved
6. Incubate at room temperature for 5 min.
7. Add 200 μl (0.2ml) chlorofame, then tightly close cap and shake vigorously with hand for
15 sec.
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8. Incubate at room temperature for 2.5 min.
9. Centrifuge to 5˚C at 11500 rpm for 15 min.
10. Transfer top aqueous layer to fresh tube (90 μl 5 times) and precipitate with 500μl
(0.5ml) of isopropyl alcohol, then vortex tubes.
11. Incubate at room temperature for 10min.
12. Centrifuge to 5˚C at 11500 rpm for 10 min.
13. Remove supernatant (will see small pellet)
14. Add 1000 μl (1ml) 70% ethanol and vortex couples of times
15. Centrifuge to 5˚C at 7200 rpm for 5 min.
16. Remove excess liquid in tubes, let it set to dry for 4 min. taking out as much liquid as
possible, YET making sure sample not completely dried.
17. Dissolve RNA with DEPC (10 μl) and mix by pipetting it
18. Add 1 μl of sample to 59 μl of DEPC water, and have control 60 μl of DEPC water (to
test concentration of sample on photometer, room N201). Remember to grab a cuvette.
a. Testing concentration on photometer
i. Make sure measurement is on RNA and not ds DNA or others
ii. Zero in with DEPC water in cuvette (make sure smooth side face light,
press dilute)
iii. Load in samples into cuvette, after taking out DEPC water (press sample)
iv. Record concentrations (greater 1.60 μg indicates completely dissolves)
b. Run on gel
i. Place UP dye into sample, centrifuge it, and vortex it.
ii. Load ladder and sample into gel (150Volts for 15 min)
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iii. Then, Acquire Image (upper left corner), filter 2 (EtBr), White (to look at),
UV on trans., Auto Exposure on, Acquire Image (upper right corner).
RNA Extraction
1. TRIzol Reagent – wear gloves
2. TRIzol Reagent – isolate total RNA from cells and tissues.
3. During homogenization/lysis, TRIzol Reagent maintains integrity of RNA; TRIzol
disrupts cells and dissolve cell components.
4. After centrifugation, add Chloroform. It separates solution into a aqueous phase and an
organic phase.
5. RNA remains in aqueous phase.
6. Aqueous phase is transferred, RNA is recovered by precipitation with isopropyl alcohol.
7. DNA is in interphase
8. Proteins in organic phase
Total RNA isolated from TRIzol Reagent is free of protein and DNA contaimination!
NOTES:
15 – 30˚C  room temperature
If using 15 ml tube  use centrifuge next door
Fast cool to 4˚C
0.3 mols | 95.53 g | 1L | 100 ml
L
| mols | 1000ml |
Revision 12-14-06