First Strand Synthesis

1. Use 20-400 |g of total cellular RNA (150 |g seems sufficient) or 1-5 |g of poly (A)+ RNA. Precipitate the RNA if necessary such that the final volume of the RNA will be 28 |L.

2. Denature the RNA by adding 5 |L of 0.1 M methylmercuric hydroxide (see Note 1) and incubating for 5 min at room temperature. Quench the reaction with 0.5 |L of 5.8 M 2-mercaptoethanol, and incubate for 5 min at room temperature.

3. The reaction mixture is then assembled as follows:

33.5 |L RNA mixture (from Subheading 3.4.1., step 2);

6.0 |L 10X first strand buffer;

6.0 |L 20 mM dNTPs containing 5-methyl-dCTP;

3.0 |L 80 mM Na-pyrophosphate;

3.0 |L placental RNAse inhibitor (30-100 U);

5.0 |L AMV reverse transcriptase (50-100 U); Incubate at 42°C for 1 h. Add 3 |L reverse transcriptase and incubate for 1 h more.

4. Determine the percent incorporation by TCA precipitation. Remove 2 pL of the reaction mix. Add 8 pL of DEPC-treated H2O, and spot 5 pL onto a Whatman GFC filter and reserve it. To the other 5 pL, add 25 pL of 2 mg/mL bovine serum albumin (BSA) and 100 pL of 20% trichloroacetic acid (TCA). Incubate on ice for 30 min, and then filter through GFC in a vacuum filtration device. Wash with 20 mL of 5% TCA, and then dry the filter for 20 min at 60°C or under a heat lamp. Transfer both filters to scintillation vials, add scintillation fluid, and count. Determine the percent incorporation of the trace label into cDNA. The yield in nanograms of cDNA is incorporation x120 (nmol each nucleotide) x4 (nucleotides) x330 (g/mol of nucleotide).

5. Dilute the cDNA to 150 pL and load onto a 1 mL Sephadex G-50 spun column in TE. Spin for 3 min at 1000 rpm, and collect the flowthrough. Measure the volume by weighing the liquid and assuming 1 g/mL for H2O. This step is necessary to remove 5-methyl-dCTP. Any remaining will be incorporated into the second strand and prevent cleavage at the 3' XhoI site.

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