John M. S. Bartlett.pdf - Bio-Nica.info

48 Pearson
3. Method
The method of Chomczynski and Sacchi (2) described in the previous protocol
works well for fixed tissues with only two modifications.
1. The extraction is initiated by incubating tissue sections or microdissected cells in 500-µL
extraction buffer for 5 min at room temperature with gentle agitation inverting several
times.
2. Add 20 µL of 2 M sodium acetate and mix gently by inversion.
3. Add 220 µL of phenol and mix gently by inversion.
4. Add 60 µL of chloroform/isoamyl alcohol (241) and vortex vigorously.
5. Place on ice for 15 min.
6. Microfuge at 12,000g for 5 min and transfer the upper phase to new microfuge tube.
7. Add 1 to 2 µL of glycogen (10 mg/mL). Glycogen is a carrier that is used if RNA quantities
are less than 1 µg. It also facilitates visualization of the pellet.
8. Add 200 µL of ice-cold isopropanol mix and store at –20°C for 30 min.
9. Microfuge at 12,000g for 15 min and discard supernatant.
10. Resuspend pellet in 200 µL of extraction buffer.
11. Repeat steps 3 through 9.
12. Wash pellet with 400 µL of cold 70% ethanol.
13. Microfuge at 12,000g for 5 min and discard supernatant.
14. Carefully remove last traces of ethanol from tube (folded sterile swab or kimwipe works
well).
15. Resuspend in 100 µL of RNAse free distilled water and incubate at 50°C for 15 min to
dissolve RNA (see Note 1).
4. Notes
1. Repeat pipetting through a narrow gauge tip can help this process.
References
1. Foss, R. D., Guha-Thakurta, N., Conran, R. M., and Gutman, P. (1994) Effects of fixative
and fixation time on the extraction and polymerase chain reaction amplification of RNA
from paraffin-embedded tissue. Diagn. Mol. Pathol. 3, 148–155.
2. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.