Competitive Rtpcr Analysis

The effect of melatonin drinking on mPerl expression in the SCN at ZT4 was examined by competitive reverse transcription-polymerase chain reaction (RT-PCR). Control mice of the ddY strain were entrained to a normal LD cycle for 10 days. The mice were then transferred to an 8 hr-advanced LD cycle 3 times with 3-day intervals from Day 2. Some mice were administered melatonin in their drinking water. On the 8 day of the treatment, the mice were sacrificed and their brains were removed and placed in ice-cold saline at ZT4. Slices (0.5 mm thick) of mice brain that contained the SCN were frozen on dry ice and the both SCN were punched out. Total RNA from the SCN (n = 4) was extracted by Trizol solution (GIBCO BRL, Gaithersburg, MD, USA). An mRNA selective PCR Kit (Takara, Osaka, Japan) was used for the reverse transcription of approximately 100 ng of RNA, and mPerl and P-actin cDNA were quantified by competitive PCR. PCR reactions were carried out for 18 and 23 cycles for p-actin and mPerl, respectively. The primer pairs used for the amplification of each product were as follows: 5'-AGG ACT CCT ATG TGG GTG ACG A-3' and 5'-CAG CCT GGA TGG CTA CGT ACA A-3' (p-actin); 5'-CAA GTG GCA ATG AGT CCA ACG G-3' and 5'-GAC ACA GGC CAG AGC CGT ACT G-3' (mPerl). The competitor DNA fragments for mPerl and P-actin was constructed with a DNA Competitor Construction Kit (Takara). The sizes of the PCR products of mPerl, mPerl competitor, P -actin and P -actin competitor were 362,292,267 and 223 bp, respectively. The PCR products were run on 3% agarose gels, and DNA in the appropriate bands were detected with an EDAS-120 system (Kodak, Rochester, USA).

The reduction of mPerl induction in the SCN after shifted-LD cycles was quantified by RT-PCR. Before this quantification, the RT products were tested for possible genomic DNA contamination. A gel analysis showed bands of expected lengths. The mPerl mRNA levels were low after shifted-LD cycle, whereas the levels were recovered in the melatonin-treated shifted mice (Figure 4). These results suggest that the phenotypic effects of melatonin treatment on the LD shift-induced decrease in locomotor activity may be mediated by the recovery of mPerl expression in the SCN.

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