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

Dual DNA/RNA Extraction 51
3. Centrifuge for 20 min at 10,000g 4°C.
4. Transfer aqueous phase to fresh tube and add 1/15 volume 7.5 M NH 4 OAc and 2 volumes
ice-cold EtOH. Incubate at –20°C for at least 1 h.
5. Centrifuge for 20 min at 10,000g, 4°C.
6. Carefully decant the supernatant and wash the pellet with 1 mL of 70% ethanol.
7. Centrifuge briefly to ensure that the pellet remains attached.
8. Carefully remove the supernatant and air dry pellet for 10 to 15 min.
9. Resuspend the DNA pellet in 50 µL of TE. Heat 5 min at 55°C then vortex thoroughly
to dissolve the DNA.
4. Notes
1. For extraction from cell lines, scrape cells into 1.5 mL of lithium chloride/urea and then
proceed from step 3 of the RNA extraction above.
2. Extraction of DNA/RNA from blood can be achieved by collecting blood in either a heparin
or EDTA containing Vacutainer by venipuncture. Store at room temperature and extract
within the same working day. Whole blood (3 mL) is placed in a 15-mL polypropylene
tube and mixed with 12 mL of red blood cell lysis solution (0.01 M Tris, pH 7.4, 320 mM
sucrose, 5 mM MgCl 2 , 1% Triton X 100). Blood is then mixed on a rolling or rotating blood
mixer for 4 min at room temperature. Lymphocytes are recovered by centrifugation at
3000g for 5 min at room temperature. Then, proceed with RNA extract at step 3 above.
3. 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.
4. 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.
References
1. Chevillard, S. (1993) A method for sequential extraction of RNA and DNA from the same
sample, specially designed for a limited supply of biological material. BioTechniques
15, 22–24.