Nusieve 3 1 agarose gel

The specificity of the PCR amplification with each primer pair was electrophoretically confirmed with a 4% NuSieve 3:1 agarose gel (Cambrex Corporation, USA). The PCR reactions were carried out in 20-μl reaction volumes using the SYBR Green PCR Master Mix (Applied Biosystems, USA) with 600 nM oligonucleotide primers and cDNA reverse-transcribed from 10 ng or 40 ng total RNA. For signal detection, the ABI Prism 7700 sequence detector (Life Technologies, CA, USA) was programmed to execute an initial step of 2 min at 50°C and 10 min at 95°C, followed by 40 thermal cycles of 15 sec at 95°C and 1 min at 60°C. The amount of the target gene was determined using a calibration curve that was constructed using serial dilutions of the target gene. The level of mRNA was expressed as the expression level relative to the average for the control group at 0 h, which was set to 1.0.

DNA was prepared from a small tail-tip biopsy taken at 21–28 days of age. The MeCP2 alleles were identified by PCR using two sets of primers. Primer set 1 (5′ primer: 5′-CAC CAC AGA AGT ACT ATG ATC-3′ and 3′ primer: 5′-CTA GGT AAG AGC TCT TGT TGA-3′) yields a product of 180 bp identifying the wild-type allele. Primer set 2 (5′ primer same as for primer set 1 and 3′ primer: 5′-ATG CTG ACA AGC TTT CTT CTA-3′) yields a product of apparent size 260 bp identifying the null allele. PCR products were electrophoresed through a 2% NuSieve 3 : 1 agarose gel (Cambrex Bio Science, Rockland, ME, USA) containing 0.5 μg/mL ethidium bromide and examined under UV light.