Application of TaqMan RT-PCR for Real-Time Semiquantitative Analysis of Gene Expression in the Striatum - Drugs of Abuse: Neurological Reviews and Protocols - page 227

allows the rapid analysis of 96 PCR samples simultaneously, without any time-
consuming downstream gel electrophoresis steps. Data are easily captured
using Sequence Detector Software (Applied Biosystems) linked to the ABI
Prism 7700, and can then be exported to Microsoft Excel worksheets for
further analysis.
The standard techniques used to generate mRNA expression data in human
and rat CNS tissues using TaqMan RT-PCR are described, including protocols
we have used for RNA extraction, cDNA synthesis, primer and probe design
and quality control, TaqMan PCR assays, and data analysis (7) .

2. Materials

1. Trizol reagent (Life Technologies).

2. Chloroform, isopropanol, and 75% ethanol.

3. RNase-free/DNase-free sterile water.

4. Poly(A) + mRNA samples (Clontech).

5. Superscript II RNase H - reverse transcriptase (200 U/ L) supplied with 5X

fi rst-strand buffer and 100 m M dithiothreitol (DTT) (Life Technologies).

µ

6. Oligo(dT) 12-18 primer (0.5 µ g/ µ L; Life Technologies).

7. 10 m M dATP, dTTP, dCTP, and dGTP (Life Technologies).

L; Life Technologies).
9. 2X TaqMan Universal Mastermix (Applied Biosystems).
10. Custom synthesized gene specifi c oligonucleotides (10

8. RNAsOUT (40 U/ µ

µ M ) and fl uorogenic

M ).
11. Genomic or plasmid DNA for standard curve generation.
12. ABI Prism 7700 or similar thermocycler with fl uorescence detection system.

TaqMan probes (5 µ

3. Methods
The methods described below outline (1) RNA extraction, (2) cDNA
synthesis, (3) primer and fl uorogenic probe design and quality control, (4)
TaqMan PCR assay, and (5) data analysis (7) .

3.1. RNA Extraction
There are several methods for extracting total RNA from tissues but we have
successfully used the Trizol method for numerous TaqMan RT-PCR studies.

1. Quickly dissect striatal tissues from rats after they are killed, wrap samples in
labeled aluminum foil, snap freeze in liquid nitrogen, and store at -80°C until
required for further processing.

2. Directly homogenize frozen rat striatal tissues in 1 mL of Trizol reagent (Life
Technologies) per 50-100 mg of tissue. Do not leave Trizol in tubes on ice, as
this greatly reduces RNA quality, and ensure the Trizol reagent has reached room
temperature after removing it from 4°C storage. Trizol aliquots can be stored