Semiquantitative Real-Time PCR for Analysis of mRNA Levels - Drugs of Abuse: Neurological Reviews and Protocols - page 212

quality is typifi ed by the Agilent 2100 Bioanalyzer Automated Analysis System
(Agilent Technologies). This instrument provides for automated analysis of
RNA with high sensitivity and small sample volume (as little as 5 ng/ µ L).

3.1.5. Protocol for Formaldehyde Denaturing Agarose Gel Electrophoresis

1. Prepare a denaturing mix: 200 µ L of formamide, 30 µ L of 10X MOPS buffer,

80 L of a 1 mg/mL solution of ethidium bromide

(exercise caution and wear gloves when handling ethidium bromide, a potent
mutagen).

µ L of formaldehyde, and 15 µ

2. Combine RNA (0.5-1.0 µ g) and nuclease-free water to a fi nal volume of 7 µ L.

Add 7 L denaturing mix, vortex-mix the sample, and place in a 65°C water

bath for 5 min.

µ

3. Remove the sample from the water bath. Add 2 µ L of 0.2% bromophenol blue

dye solution.

4. Combine 36.5 mL of nuclease-free water, 0.5 g of agarose, and 5.0 mL of a 10X
MOPS buffer solution. Heat in a microwave or on a hot plate until the agarose
is completely in solution.

5. In a fume hood, add 8.5 mL of formaldehyde, pour molten solution into a gel
manifold, insert comb, and allow gel to solidify (approx 30 min). Assemble
in a submarine.

6. Format with the electrophoresis apparatus.

7. Load the entire sample (16 L) onto 1% denaturing agarose gel and resolve in a

1X MOPS buffer solution at 50 V for 30-60 min.

µ

8. Visualize bands under UV light in a UV light box. Prepare a photographic image
(Polaroid or gel-documentation workstation.

3.2. cDNA Production
To eliminate potential DNA contamination in the RNA preparation, total
RNA can be treated with DNase I (0.4 U/µg of RNA) according to the reagent
supplier's instructions. Alternatively, in lieu of DNase I treatment, and/or as an
additional control, it is advisable to add a “no reverse transcriptase” reaction
to the experiment. To accomplish this, simply prepare one reaction tube or
well exactly as the others, but leave out the RT enzyme. Subsequently, use the
resulting “reaction product” for PCR (no PCR product = no genomic DNA
contamination). When performing quantitative or semiquantitative PCR, it
is important to rule out contamination of the RNA preparations with geno-
mic DNA.

3.2.1. Priming for First-Strand Synthesis
There are three types of oligonucleotide priming protocols used with the
RT reaction. The method of choice, in our experience, is gene-specifi c primers
( see Note 4 ). If the primer set is well designed and working optimally, the PCR