DNA, RNA and protein extraction from skin tissue

Effective, efficient sample preparation is critical to successful downstream results. Now an effective method exists for the extraction of intact, biologically functional macromolecules from skin.

Animal and human tissues can be the toughest samples to isolate high-quality DNA, RNA and proteins. However, using the FastDNA Spin Kit and FastRNA Pro Green Kit in combination with the FastPrep instrument, full homogenisation of any sample including bone and tumours, and more elastic samples like skin, can be achieved within a few seconds. This method saves hours of work during sample preparation and provides high yields of DNA, RNA and proteins.

RNA and protein extraction from skin tissue

The FastPrep and associated matrices have demonstrated successful lysis and dual extraction of RNA and proteins from skin tissue in three runs of 40 seconds each.

Materials

• FastPrep instrument
• Lysing Matrix D tubes
• Sample: Human skin biopsies from a 3 mm punch (weighing 19 mg on average)

Protocol and parameters

1. Add the skin sample to a Lysing Matrix D tube.
2. Add 1 mL of a guanidine thiocyanate lysis buffer (5.1 M guanidine thiocyanate, 50 mM sodium citrate, 50 mM EDTA, 0.5% ß-mercaptoethanol).
3. Homogenise in the FastPrep instrument for 3 x 40 seconds at a speed setting of 6.0. Place the tubes on ice for 5 minutes between each run.
4. Centrifuge at 14,000 x g for 5-10 minutes to pellet debris.
5. Proceed with the RNA and protein extraction protocol.

Results

The average yield of 1.4 μg RNA obtained with the FastPrep System was 57% higher than yields obtained with the Polytron, while the average yield of 170 μg protein obtained with the FastPrep System was 53% higher than yields obtained with the Polytron.

To verify the high-quality nature of the RNA, samples were analysed with the Agilent 2100 Bioanalyzer. Samples had ribosomal integrity number numbers of between 8.4 and 8.9, which is consistent with high-quality RNA (see figure).

The RNA was run on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, Calif.) using the RNA 6000 Pico LabChip kit to determine the quality of the samples. The 28S and 18S ribosomal bands show a greater than 2:1 ratio, and the calculated RNA ribosomal integrity numbers of the samples ranged from 8.4 to 8.9, verifying high-quality RNA. Shown above are the gel images for 11 RNA samples and below are two representative electrophoretic graphs showing the RNA peaks.

The quality of extracted proteins was assessed by two-dimensional gel and Western blot analysis. There was distinct spot resolution and sufficient protein isolated from a single biopsy to produce five to six two-dimensional gels. For Western blotting, a primary antibody against GADD-45 was used to probe the membrane. GADD-45 antibody detects both the alpha and beta portions of the protein, although it is more sensitive for the alpha portion.

The FastDNA Spin Kit is used with the FastPrep instrument to lyse and subsequently isolate DNA from up to 200 mg of almost any sample in less than 30 minutes.

The FastRNA Pro Green Kit is designed to efficiently isolate total RNA from any type of plant and animal tissue or cultured cells.

Visit www.mpSamplePrep.com to access articles and other educational materials highlighting successful sample preparation using the MP Biomedicals product line.