Analysis of the Products

The product of the amplification may contain several bands that migrate at the same position in the sequencing gel. The best way to assess the identity of the bands in the first place is to clone the PCR product into a suitable vector for cloning PCR products. Several clones should be sequenced to verify homogeneity of the initial product. Once a major clone has been identified, Northern blot hybridization should be performed using the cloned major PCR product as a probe. We do not recommend either dot-blot or Southern blot hybridization of the PCR products with a hot cDNA probe to verify positive clones, since repetitive sequences and labeled ribosomal RNA can give rise to nonspecific positive signals.

3.3.1. Cloning and Sequencing of the PCR Products

Because a single size band may contain several DNA species, several clones should be sequenced so that the real differentially displayed clones are not missed. If several sequences appear, individual clones should be tested as probes in Northern blot analysis, as described below. An alternative strategy has been described to clone specific products after Northern blot hybridization. This procedure involves the capturing and the reamplification of the hybridized cDNA from the membrane (13).

Several kits are available to clone PCR products. The TA cloning kit from Invitrogen (San Diego, CA) allows direct cloning of homogeneous PCR products without purification and/or blunting of the DNA ends. If this kit is used, take 2 or 3 |L of the PCR mixture (after verification that a sufficient amount of the desired band is present) for the ligation reaction. Do not dry concentrate the product since dNTPs can inhibit the activity of T4 DNA ligase. An alternative kit form Stratagene requires blunt-ending of the cDNA prior to ligation. Both manufacturers provide excellent step-by-step protocols for the use of their products, so we will not provide a protocol for this procedure.

After cloning, clones should be sequenced for further analysis. This can be performed by using the dideoxy chain termination method using primers flanking the insert sequence (T3 and/or T7 primers) as initiators for the polymerase. All reagents required for sequencing can be purchased from USB (Cleveland, OH) as a kit, accompanied by instructions from the manufacturer.

3.3.2. Northern Hybridization Labeling of the Probe

Approximately 25 ng of the PCR product should be labeled with random hexanucleotides according to the procedure of Feinberg and Vogelstein (12). A detailed procedure including the solutions needed, can be found in Chapter 37 (Subheading 3.9.), and also Note 7 in this chapter. Adding 10 pmol of the random oligonucleotide used for amplification to the labeling mix containing the random hexamers will help to reach a better specific activity.

3.3.3. Electrophoresis of RNA

1. Preparation of the gel: Add 1 g of agarose to 72.6 mL dH2O. Melt in a microwave oven. Once the flask can be handled (55-60°C), add 10 mL of 10X MOPS buffer and 17.4 mL of 37% formaldehyde. Assemble a horizontal gel cast in a fume hood, because formaldehyde vapors are toxic. Allow the gel to solidify.

2. Preparation and running of samples: From the original RNA solutions made, resuspend 10-20 mg of RNA in 7 |L of nuclease free water. Then add 15 |L deionized formamide, 3 |L 10X MOPS, 5 |L 37% formaldehyde, and 1 |L of a 1 mg/mL ethidium bromide solution. Heat at 65°C for 5-10 min. Chill on ice, add 7 |L of 6X gel loading buffer, and load on the gel that should be in a gel apparatus in 1X MOPS running buffer. Run at 5.6 V/cm (distance between electrodes), until the bromophenol blue is at 3 cm from the bottom of the gel.

3.3.4. Blotting and Hybridization

Wash the gel twice for 30 min each time in 10 mM phosphate buffer, pH 6.8. To make this solution, mix 51 mL of 0.5 M NaH2PO4 with 49 mL of 0.5 M Na2HPO4. Dilute the resulting 0.5 M solution to 10 mM. Place the gel in a blotting apparatus, and transfer to a nylon membrane in 20X SSC. Once transfer is completed, check the position of the ribosomal markers with a handheld UV lamp (sometimes they are visible without it), and mark their position with a soft pencil. Also mark the orientation of the gel and the position of the wells. Wash the membrane in 2X SSC and fix the RNA in a UV crosslinker

(Stratalinker or similar) or in an 80°C oven for 1-2 h. Prehybridize the gel in a solution containing 50% formamide, 5X Denhardt's solution, 0.1% SDS, 20 mM phosphate buffer, pH 7.0, 5X SSC, and 250 |g/mL of denatured salmon sperm DNA (16) at 42°C for at least 1 h. To denature the DNA, heat it at 100°C for 10 min, and immediately chill on ice. After prehybridization, discard the solution, and add fresh solution containing the same ingredients plus 1-5 x 106 cpm/mL of probe. Remember that the probe has to be denatured prior to adding it to the hybridization solution. Leave hybridizing overnight at 42°C.

3.3.5. Washings and Autoradiography

Wash the membrane twice in the following solutions: 2X SSC/0.1% SDS at room temperature for at least 15 min each; 0.1X SSC/0.1% SDS at 42°C for at least 30 min each; same solution at 50°C for at least 30 min each. Check the extent of washing with a Geiger counter. If the background is high on the membrane (high counts where no RNA is expected), repeat washing with the same solution at 55 °C for at least 30 min each. If the background is still high, wash briefly several times in the same solution at 60°C. Once washed, wrap the wet membrane in Saran Wrap, and expose to double-side emulsioned autoradiography film at -80°C using an intensifying screen. Sometimes bands require several days of exposure to be seen (15). Once bands are detected, the size of the mRNAs can be roughly calculated as they migrate according to the logarithm of their molecular weight. The known sizes to draw a curve should be the 28 and 18S ribosomal RNAs whose position was marked with a pencil (16).

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