Advanced Concepts

Specialized collection tubes are available for the isolation of RNA from whole blood (e.g., from Qiagen or Applied Biosystems). These tubes contain proprietary reagents that stabilize the intracellular RNA for several days at room temperature and longer at refrigerator temperature. The RNA can be isolated on a solid matrix.

cytes in blood and bone marrow samples are lysed by osmosis or separated from WBCs by centrifugation. When dissociating tissue, the sample should be kept frozen in liquid nitrogen or immersed in buffer that will inactivate intracellular RNAses. This is especially true for tissues such as pancreas that contain large amounts of innate RNAses. Bacterial and fungal RNA are also isolated by chemical lysis or by grinding in liquid nitrogen. Viral RNA can be isolated directly from serum or other cell-free fluids by means of specially formulated spin columns or beads. As most total RNA isolation methods cannot distinguish between RNA from microorganisms and those from host cells, cell-free material should be used for these isolations.

The cell lysis step for RNA isolation is done in detergent or phenol in the presence of high salt (0.2-0.5 M NaCl) or RNAse inhibitors. Guanidine thiocyanate is a strong denaturant of RNAses and can be used instead of high salt buffers. Strong reducing agents such as 2-mercaptoethanol can also be added during this step.

Once the cells are lysed, proteins can be extracted with phenol (Fig. 4-5). Acid phenol:chloroform:isoamyl alcohol (25:24:1) solution efficiently extracts RNA. Chloroform enhances the extraction of the nucleic acid by denaturing proteins and promoting phase separation. Isoamyl alcohol prevents foaming. For RNA, the organic phase must be acidic (ph 4-5). The acidity of the organic phase can be adjusted by overlaying it with buffer of the appropriate pH. In some isolation procedures, DNAse is added at the lysis step to eliminate contaminating DNA. Alternatively, RNAse-free DNAse also may be added directly to the isolated RNA at the end of the procedure. After phase separation, the upper aqueous phase containing the RNA is removed to a clean tube, and the RNA is precipitated by addition of two volumes of ethanol or one volume of isopropanol. Glycogen or yeast-transfer RNA may be added at this step as a carrier to aid RNA pellet formation. The RNA precipitate is then washed in 70% ethanol and resuspended in RNF buffer or water.

Solid-phase separation of RNA begins with similar steps as described above for organic extraction. The strong denaturing buffer conditions must be adjusted before application of the lysate to the column (Fig. 4-6). In some procedures, ethanol is added at this point. Some systems provide a filter column to remove particulate material before application to the adsorption column. As with DNA columns, commercial reagents are supplied with the columns to optimize RNA adsorption and washing on the silica-based matrix.

The lysate is applied to a column in high-salt chaotropic buffer, and the adsorbed RNA is washed with supplied buffers. DNAse can be added directly to the adsorbed RNA on the column to remove contaminating DNA. Washing solutions and the eluant can be drawn through the column by gravity, vacuum, or centrifugal force. Small columns of silica-based material that fit

Cells in suspension

Cells in suspension

Lysis

(guanidinium isothiocyanate)

Lysed cells

■ Figure 4-5 Organic extraction of total RNA.

Lysis

(guanidinium isothiocyanate)

Lysed cells

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