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

56 McDonagh
2. Add 0.4 mL of TNE buffer with SDS, proteinase K, and poly A to the extraction tubes.
3. Add 100 µL of concentrated plasma or serum and mix immediately.
4. Incubate the lysates for 1.5 to 2 h in 37°C water bath.
5. Phenol extraction: Add 450 µL of phenol to the extraction tubes. Mix extensively and
centrifuge at 13,000g for 10 min.
6. Phenol/chloroform extraction: Transfer the upper aqueous layer to a fresh tube and add
0.45 mL of phenolchloroform (11). Vortex and centrifuge as above.
7. Chloroform/isoamylalcohol extraction: Transfer the upper aqueous layer to a fresh tube
and add chloroformisoamylalcohol (501). Vortex and centrifuge as above.
8. Ethanol precipitation: Transfer the aqueous layer to a fresh tube containing 40 µL of 3 M
Na-acetate, pH 5.2, and 800 µL of ethanol. Mix and precipitate the nucleic acids at –20°C
overnight or at –70°C for 30 min.
9. Collect the nucleic acid by centrifugation at 15,000g for 20 min at 0°C.
10. Discard the supernatant and wash the pellet with 1 mL 80% (v/v) ethanol.
11. Dry the precipitate on a dry block and dissolve in 25 µL of RNase-free water. These
extracts can be stored at –70°C.
4. Notes
1. It is important to process the samples alongside any controls and standards to allow
for the loss of RNA through sample preparation methods. This is especially important
when preparing template for a limiting dilution curve or when using an external curve
dilution series.
2. Although several commercial extraction systems have become available, we have not
attained similar levels of sensitivity as with the protocol suggested here. However,
extraction systems using guanidinium thiocyanate alongside phenol/chloroform or silica
have been successfully used in ultrasensitive assays.
References
1. Clementi, M., Menzo, S., Bagnarelli, P., Manzin, A., Valenza, A., and Varaldo, P. E. (1993)
Quantitative PCR and RT-PCR in virology. PCR Meth. Appl. 2, 191–196.