Molecular Techniques and Methods
Isolation of RNA Using RNAzol
Copy Right © 2001/ Institute of Molecular Development LLC
MATERIALS AND SOLUTIONS
1. Determine the approximate weight of any tissue samples to be studied.
2. Distribute RNAzol to polypropylene tubes on the basis of 2 ml of RNAzol per 100 mg of tissue.
The RNAzol should be kept cold and protected from light; take care when handling the RNAzol because it is highly caustic.
3. Place the tissue sample in RNAzol and homogenize it thoroughly.
4. Snap-freeze the sample in liquid nitrogen and store at -70
C for future extraction.
5. Thaw frozen homogenate for approximately 5 minutes in a 37
C water bath.
6. Add 0.2 ml of a 24:1 mixture of chloroform: isoamyl alcohol for every 2 ml of homogenate and shake the samples vigorously for 15 seconds.
Let the samples sit on ice for 5 minutes.
7. Centrifuge the samples at 12,000
for 15 minutes at 4
8. Carefully remove the aqueous (top) phase that contains the RNA and transfer it to another tube; store on ice.
Care must be taken to avoid the white interphase layer.
9. To each sample, add a volume of cold isopropanol that is equal to the volume of the aqueous phase.
10. Mix the tubes gently and store the samples on ice.
11. Centrifuge the samples at 12,000
for 15 minutes at 4
The RNA should form a whitish/yellow pellet at the bottom of the tube.
12. Carefully decant the isopropanol.
13. Wash the RNA pellet by adding one volume of cold 75% ethanol, and resuspend the RNA pellet by shaking or pipetting.
14. Centrifuge the sample at 12,000
for 8 minutes at 4
15. Carefully decant the ethanol and dry the pellets by air or under vacuum.
Vacuum drying is more expedient, but take care not to overdry the pellet.
16. Solubilize the RNA pellets in 20-50 ul of
It is usually helpful to freeze and then thaw the samples to increase solubility.
17. Quantify the product spectrophotometrically by measuring the absorbance at 260 nm of an aliquot.
The 260/280 ratio should be 1.8 or above. If the ratio is low, the sample should be reextracted.
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