Alcidine Orange Cell Staining

Checking Rna Integrity Agarose Gel

Fig. 5. RNA integrity gel. RNA was isolated from cultured rat liver epithelial cells by the acidic phenol extraction method. Two micrograms of RNA per sample were run on an integrity gel for 2 h at 90 V. Lane A contains an RNA molecular size standard. Lanes B and C contain intact RNA (rRNA bands are clearly visible), whereas the samples in lanes D and E are degraded. The RNA in lane D was prepared in a small volume of tap water (instead of RNAase-treated water) and the RNA in lane E came in contact with human skin during sample preparation.

Fig. 5. RNA integrity gel. RNA was isolated from cultured rat liver epithelial cells by the acidic phenol extraction method. Two micrograms of RNA per sample were run on an integrity gel for 2 h at 90 V. Lane A contains an RNA molecular size standard. Lanes B and C contain intact RNA (rRNA bands are clearly visible), whereas the samples in lanes D and E are degraded. The RNA in lane D was prepared in a small volume of tap water (instead of RNAase-treated water) and the RNA in lane E came in contact with human skin during sample preparation.

information obtained from visualization of the RNA often outweighs the inconvenience of a reduced signal. Alcidine orange (25) and Stains All (a cationic carbocyanine dye) (25,27) are alternative dye choices for staining and visualizing the RNA.

The recommended buffer for electrophoresis of RNA contains 3-(N-morpholino)propanesulfonic acid (MOPS) (6). A 5X or 10X stock of MOPS buffer (see Table 2) should be prepared in DEPC-treated water, brought to a pH of 7.0, filter-sterilized, and stored at room temperature in a dark bottle. Autoclaving or exposure to light can cause yellowing of the buffer. Although pale yellow buffer could still be used, buffer exhibiting a darker shade of yellow should not be used. Constant circulation of buffer during electrophoresis in the same direction as electrical current prevents accumulation of buffer components and formaldehyde in the positive buffer chamber. The general rule for running RNA gels is 3-4 V/ cm length. The bromophenol blue (in the RNA sample buffer) should migrate at least 8 cm before electrophoresis is terminated. Because there is no reliable storage method for elec-trophoresed RNA gels, it is optimal to transfer the nucleic acids to a solid support immediately after electrophoresis. For this reason, many workers choose to run gels overnight at a low voltage (20-30 V).

If ethidium bromide is not included in the gel, the lane containing RNA standards must be excised and stained. The distance from the loading well to each band should be measured to generate a standard curve. A plot of the log10 of the RNA fragment sizes against distance migrated provides a curve by which sizes of RNA species detected by hybridiza tion can be calculated. Prior to transfer of the RNA to a nitrocellulose or nylon membrane, the formaldehyde should be removed from the gel. This is typically accomplished by soaking the gel in several changes of DEPC-treated water or transfer buffer.

9.3. TRANSFER OF RNA TO A SOLID SUPPORT For optimal results, the electrophoresed RNA should be transferred to a nylon membrane. The methods of transfer (capillary, vacuum, and electrophoretic) are the same as for DNA transfer and are discussed in previous sections. Because the RNA molecules are already single-stranded, there is no need to denature the gel prior to transfer. However, both the gel and the nylon membrane should be equilibrated in the appropriate transfer buffer (see Table 2) prior to transfer. Traditional capillary transfer works well for most Northern blots, although complete transfer takes 12-18 h (see Fig. 3). When time is limited, the downward alkaline transfer or vacuum transfer method can be used. Electrophoretic transfer is ideal when the target mRNA is present in a very low quantity or when large RNAs need to be transferred. When fixing a Northern blot, experimental evidence indicates that fixation by UV irradiation is superior to baking at 80°C (21). As with DNA, overirradiation can cause extensive crosslinking of the RNA and diminish the final signal, so optimal UV exposure should be determined empirically. Irradiated, air-dried membranes can be stored at room temperature until they are ready to be hybridized to a nucleic acid probe.

9.4. INTERPRETATION OF THE NORTHERN BLOT When analyzing any experimental sample by Northern blot, it is recommended to include a control on the gel consisting of

Table 3 Northern Blot Analysis

1. Preparation of RNA samples a. Combine an appropriate quantity of purified total RNA or mRNA with 2.0 ||L of 5X MOPS buffer, 3.5 ||L of 37% formaldehyde, pH > 4.0, 10.0 |L formamide, and RNAase-free water to give a total volume of 20 |L.

b. Add 4.0 |L of RNA loading dye (Table 2) and heat samples to 65°C for 10 min. Open the caps for 5-10 min prior to loading the samples to allow traces of ethanol to evaporate.

2. Preparation of the formaldehyde gel a. Melt agarose (final concentration should be 1.2%) in RNAase-free water by boiling in a microwave. Cool to 60°C, add 5X MOPS

gel buffer (1/5 vol) and 37% formaldehyde (1/5.6 final volume). If desired, ethidium bromide can be added to the gel (0.5 |g/mL).

b. Pour gel immediately into clean, level gel tray and insert the appropriate sample comb(s).

c. When the gel has solidified (approx 20 min), place it in the electrophoresis tank, remove the sample comb, and fill the tank with 1X

MOPS buffer until the liquid covers the gel by 3-4 mm. Prerun the gel for 5 min at 5 V/cm length.

3. Running the gel a. Load the RNA samples carefully into the sample wells. Reserve one well for loading appropriate RNA size standard (1-3 |g are usually sufficient). If the samples "float" out of the well as they are loaded, they might still contain traces of ethanol. Reheating the samples briefly (uncapped) will eliminate this problem.

b. The gel may be run overnight at 20-30 V, or at 3-4 V/cm length. Electrophoresis should continue until the bromophenol blue tracking dye has migrated a minimum of 8.0 cm.

c. Once the samples have entered the gel (approx 20 min), begin circulating the buffer with a peristaltic pump in the same direction as the electrical flow.

d. When electrophoresis is complete, record the migration distances of the RNA ladder (if ethidium bromide was not added to the gel, this lane should be cut away and stained in a 2-| g/mL solution of ethidium bromide). If ethidium bromide was added to the gel, the entire gel should be visualized with a UV light source and photographed. Remove the lane containing the RNA standards prior to transfer.

4. Capillary transfer of the RNA to a nylon membrane a. Cut a piece of nylon membrane to the size of the gel and prewet it in RNAase-free water. Equilibrate the membrane in alkaline transfer buffer for 15 min.

b. Remove the formaldehyde from the gel by soaking 3X 10 min in RNAase-free water.

c. Assemble the transfer setup as shown in Fig. 3 and allow the RNA to transfer for 1-3 h.

d. Air-dry the nylon membrane completely; or air-dry the membrane briefly than crosslink with UV irradiation.

5. Hybridization with nucleic acid probe a. Prehybridize the membrane for at least 1 h at 42°C in 10-20 mL of prehybridization solution.

b. Prepare the labeled probe as per manufacturer's instructions.

c. Replace prehybridization solution with hybridization solution containing the labeled probe and incubate 12-14 h at 42°C.

6. Washing the hybridized membrane a. Wash the membrane 2X 15 min in 2X SSPE, 0.1% SDS at 42°C, and then 2X 15 min in 1X SSPE, 0.1% SDS at 42°C.

b. If a radioactive probe was used, check the membrane with a Geiger counter. If additional washing is needed, wash 2X 15 min in 0.5X SSPE, 0.1% SDS at 42°C.

7. Visualization of hybridized probe (radiolabeled probes): Wick excess buffer from the washed membrane and wrap the filter in plastic wrap;

expose the membrane to film in a lighttight cassette at -70°C. Develop the autoradiograph after 24 h, adjusting the exposure time as needed.

RNA isolated from a control cell or tissue. Interpretation of a Northern blot is most accurate when actual numerical values are assigned to the bands present on the final blot, which represent a positive signal. Colorimetric detection methods are the least sensitive and should be avoided if comparisons are to be made between two test samples in which differences in expression might be subtle. Both chemiluminescent and radioactive probes can be exposed to X-ray film to produce an autoradiograph. Figure 6 shows an autoradiograph generated from a Northern blot that was probed with a 32P-labeled cDNA probe. The autoradiograph can be analyzed by a scanning densitometer, which measures the density of each band and assigns a numerical value to the band. This analysis allows the detection of very small variations in expression. However, for interpretation to be accurate, a control must be included for the amount of mRNA that is present in the samples being compared. Some researchers rely on ethidium bromide staining for the gel prior to transfer and hybridization. However, visualization of the mRNA in formaldehyde-

containing gels is difficult and not quantitative. A more reliable method is to perform a second hybridization reaction with the membrane, utilizing a probe (labeled in the same way as the probe used to detect the gene of interest) for a "housekeeping gene," such as actin (a structural protein) or cyclophilin (cyclosporine-binding protein). Numerical values are then generated for the positive signal from the housekeeping gene via scanning densitometry. The expression of the gene of interest is then expressed as a function of the expression of the housekeeping gene, thereby "normalizing" each signal relative to the actual quantity of mRNA present in that particular lane of the gel. If radiolabeled or chemiluminescent probes are used, the blots can be analyzed directly in a phos-phorimager, which detects positive signals on the blot and expresses the value as actual counts per minute of radioactivity. This instrument is particularly useful in detecting very weak positive signals and can often shorten the time required to detect positive bands compared to generating traditional autoradiographs.

Fig. 6. Northern blot analysis of the urokinas plasminogen activator (uPA) mRNA. RNA was isolated from nine different rat liver epithelial cell lines by acidic phenol extraction and was subsequently purified by binding to oligo-dT. Two-microgram samples of mRNA from each cell line were subjected to Northern blot analysis as described in Table 3. The radiolabeled probes were generated by random primer extension utilizing a 1.5-kb fragment of the uPA gene and a 1.2-kb fragment of the cyclophilin gene. The blot was first probed with the probe for uPA and an autoradiograph was generated. Then, the membrane was stripped in a solution containing 50% formamide and 2X SSPE and reprobed for the cyclophilin mRNA. Although the expression of the "housekeeping" gene (cyclophilin) remains relatively constant among the samples, the expression of the 2.4-kb uPA mRNA varies greatly among this group of cell lines.

Fig. 6. Northern blot analysis of the urokinas plasminogen activator (uPA) mRNA. RNA was isolated from nine different rat liver epithelial cell lines by acidic phenol extraction and was subsequently purified by binding to oligo-dT. Two-microgram samples of mRNA from each cell line were subjected to Northern blot analysis as described in Table 3. The radiolabeled probes were generated by random primer extension utilizing a 1.5-kb fragment of the uPA gene and a 1.2-kb fragment of the cyclophilin gene. The blot was first probed with the probe for uPA and an autoradiograph was generated. Then, the membrane was stripped in a solution containing 50% formamide and 2X SSPE and reprobed for the cyclophilin mRNA. Although the expression of the "housekeeping" gene (cyclophilin) remains relatively constant among the samples, the expression of the 2.4-kb uPA mRNA varies greatly among this group of cell lines.

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