Semiquantitative Real-Time PCR for Analysis of mRNA Levels - Drugs of Abuse: Neurological Reviews and Protocols - page 209

2.2. Equipment and Supplies
1. Agarose gel apparatus and power supply.
2. Light box (with UV lamp).
3. Gel-documentation workstation (Alpha Innotech, San Leandro. CA).
4. Spectrophotometer (with UV lamp).
5. Thermocycler (for reverse transcription reactions).
6. Water bath(s).
7. Real-time PCR thermocycler. Some examples: ABI Prism ® 7000 Sequence
Detection System (Applied Biosystems, Foster City, CA), iCycler (Bio-Rad,
Hercules, CA), Smart Cycler System (Cephid, Sunnyvale, CA), or LightCycler
System (Roche Molecular Biochemicals Indianapolis, IN).
2.3. Software
1. Adobe Photoshop Image capture (San Jose CA).
2. RNA quantitation (TINA, Raytest, Wilmington, NC; helpful but not absolutely
necessary).
3. Primer design: ABI Prism® Primer Express ™ from Applied Biosystems; or
Primer3 from the Whitehead Institute at MIT (http://www.genome.wi.mit.edu/
genome_software/other/primer3.html).
4. Kinetic real-time PCR sequence detection and analysis (ABI Prism® 7000
supplied by Applied Biosystems).
3. Methods
The methods outlined in this subheading describe a typical experimental
series using RNA isolated from brain regions of experimental animals, and
then assayed using the ABI 7000 thermocycler (other real-time instruments,
e.g., ABI's 7700, the Bio-Rad iCycler, or Roche LightCycler can also be used
for kinetic real-time PCR). This particular platform is chosen for discussion
because the authors have this instrument on-site, and have experience of using
it ( see Note 2 ).
3.1. RNA Preparation
The single most important factor in the ability to generate quality results
from a quantitative real-time PCR experiment, assuming a valid experimental
design for the generation of “control” and “treated” samples, is the preparation
and use of high-quality RNA. Total RNA (or mRNA) can be reliably and
repeatedly generated from any number of biological samples including whole
blood, tissues (e.g., brain, liver, etc.), and cells in culture. In this subheading,
we discuss the isolation and assay of total RNA from brain tissue.