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

46 Bartlett
3. Methods
3.1. RNA Extraction
1. Tissues should ideally be collected fresh and stored in liquid nitrogen. Routinely samples
are collected on ice and transported for freezing within 30 to 60 min.
2. Tissues are disaggregated using a Braun-micro dismembranator. Teflon vessels and steel
ball bearings are cooled in liquid nitrogen before use. Frozen tissue (50–500 mg) is placed
in the vessel with a single ball bearing and agitated at 1000 cycles/second for 60 s. The
vessel is then re-cooled in liquid nitrogen. This process is repeated until tissue is powdered
(usually 2×; see Note 1).
3. Immediately after disaggregation of tissue, tissue material is resuspended while frozen
by adding 1.5 mL of LiCl/Urea and transfer to a separate tube. The vessel is washed a
further 2× with 1.5 mL of LiCl/Urea and the washing combined with the original sample.
The resuspended medium is made up to 6 mL in LiCl/Urea and sonicated for 2× 30 s
at maximum power using a probe sonicator. The sonicated samples are stored overnight
at 4°C (see Note 2).
4. Centrifuge at 15,000g, 4°C for 30 min. The supernatant is discarded and the pellet washed
with a further 6 mL of lithium chloride/urea, recentrifuged (15,000g, 4°C for 30 min) and
the supernatant again discarded.
5. The pellet is resuspended in 6 mL of Tris-HCl/SDS with 50 µg/mL proteinase K (Boehringer
Mannheim, UK) and incubated at 37°C for 20 min.
6. Samples are extracted with 100% phenol, followed by phenol:chloroform:isoamyl-alcohol.
After each extraction, the sample is centrifuged at 2000g at room temperature for 10 min
and the aqueous phase recovered.
7. After the final extraction, 300 µL of 8 M LiCl and 2.5 volumes absolute alcohol are added
and samples stored at –20°C for 30 min overnight. RNA is pelleted by centrifugation
at 4000g, 4°C for 45 min. The supernatant is discarded and the RNA pelleted dried and
resuspended in DEPC-treated distilled water. Concentrations are estimated by optical
density at 260/280 nm.
4. Notes
1. Disaggregation is critically dependent on tissue structure. Most tissues are readily
disaggregated in two 60-s bursts. Other tissue types (e.g., fibrous tissues) may require
longer periods to disrupt tissue. If a mechanical dismembranator is not available, other
methods of tissue homogenization work equally well, either using a mortar and pestle
or blade homogenizers.
2. Other methods can be used to lyse cells, such as passage through a syringe needle, etc.
Extraction of RNA from solid tissues can be problematic because many of the commercial
systems available for RNA extraction are validated for extraction of RNA from cell
culture material or blood lymphocytes. These kits have often been less successful with
tissue-derived material.