Protective Effect of Taurine on the Decreased Biogenic Amine Neurotransmitter Levels in the Brain of Mice Exposed to Arsenic - Advances in Experimental Medicine and Biology - page 263

perchloric acid (0.1 mol/L) and centrifuging twice at 10,000 × r/min for 10 min at
4°C. Before detection, the supernatant was collected and filtered through 0.45 m m
Acrodisc filter. Each sample (20 ml) was injected into an HPLC system (Waters
1525) with fluorescence detector (EICOM, Kyoto, Japan) with an ODS column
(EICOMPAC SC-5 3.0 mm × 150 mm; EICOM Inc., Kyoto, Japan). The mobile
phase was sodium acetate 0.1 mm/L EDTA·2Na (pH 5.0)/methanol (95%/5%). The
flow rate was kept constant at 1.0 ml/min and the temperature of the column was
25°C. The levels of NE, DA, and 5-HT were measured. Each of the standard solu-
tions of the three monoamine neurotransmitters was prepared at a concentration of
0.1 ng/ml. Each standard (20 ml) was analyzed by HPLC, and the standard chro-
matographic peaks per 1 ng for each sample were obtained. The amount of each
monoamine was determined with peak-area ratios using HPLC chromatogram
analysis software, eDAQ Power Chrom (eDAQ, New South Wales, Australia).

26.2.5 Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from the brain parts of each mouse in each group using
RNAiso Plus reagent (TaKaRa) according to the instructions of the manufacture.
The yield of total RNA was determined by measuring the absorption at 260 and
280 nm separately, and 500 ng of which was in reverse transcribed to first strand
cDNA in a 10 ml reaction volume. Reverse transcription was performed using the
PrimeScript ® RT Master Mix Perfect Real Time bought from TaKaRa (Dalian,
China) according to the instructions. Briefly, 2 ml 5× PrimeScript ® RT Master
Mix with 3 ml RNase-free water and 5 ml total RNA was added in the reaction
system. And the mixture was incubated at 37°C for 15 min. Then the reaction
was stopped by heating at 85°C for 5 s. The solution for cDNA was stored at 4°C
until use.

26.2.6 Real-Time Reverse Transcription-Polymerase

ChainReaction

Real-time reverse transcription-polymerase chain reaction (Real-Time RT-PCR)
was performed according to the SYBR ® Premix Ex TaqTM kit bought from TaKaRa
(Dalian, China). The primers are based on the sequences from NCBI database. The
primers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tyrosine hydrox-
ylase (TH), tryptophan hydroxylase (TPH), and dopamine-b -hydroxylase (DBH)
are designed by TaKaRa Biotechnology Company (Japan), and the related impor-
tant information of these primers is presented in Table 26.1 . The PCR programmer
consisted of an initial denaturation at 95°C for 30 s, followed by 40 PCR cycles:
95°C for 5 s and 60°C for 30 s with Thermal Cycler Dice ® Real Time System