First strand cdna synthesis kit

Messenger RNA (mRNA) was extracted from the dorsal skin of mice at 12 weeks of age using
the QuickPrep Micro mRNA Purification Kit (Amersham Biosciences, Buckinghamshire,
England). First-strand complementary DNA (cDNA) was synthesized using a First-Strand cDNA
Synthesis Kit (Amersham Biosciences). A cDNA fragment for the Oca2 gene
was then amplified by PCR using 2 sets of primers (Table 1). These primers were designed based on the reported sequence for the
mouse Oca2 (NM_021879). The primer set 1 could amplify a region that
contains the nonsense substitution in NCT. The primer set 2 was designed to amplify a
downstream region of the nonsense substitution. PCR amplification was performed using
TaKaRa LA Taq DNA polymerase in 25 µl reactions
according to the manufacturer’s instructions. PCR reactions were carried out using the
following conditions: initial denaturation at 94°C for 1 min, followed by 40 cycles at
94°C for 20 s, 55°C for 15 s, and 72°C for 1 min. A 10 µl aliquot of each
PCR product was subjected to agarose gel electrophoresis. The PCR products were purified
and directly sequenced with the primers.

Total RNA was isolated from cell lines with TRIzol (Invitrogen) and cDNA was prepared from 2 mg of RNA using a First-Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Amersham Biosciences, Amersham, UK). Oligonucleotide sequences were as follows: SHP-1, 5′-GCC CAG TTC ATT GAA ACC AC- 3′ (sense) and 5′-GAGGGAACCCTTGCTCTTCT-3′ (antisense); GAPDH, 5′-CGACCACTTTGTCAAGCTCA-3′(sense) and 5′-AGGGGTCTACATGGCAAC TG-3′ (antisense); RFX-1: 5′-CGGCAAGCACCAGCTACTAC-3′ (sense) and 5′-GGACACGTACATGGGCATGG-3′ (antisense). For QPCR, thermocycling was performed in a final volume of 20 μl containing 2.5 μl of cDNA sample, 200 nM of each of the primers, and 6.5 μl of SYBR Green I Master Mix (Roche) with Roche LightCycler 480 sequence detection system (RocheApplued Science, Foster, California). The following PCR conditions were used: denaturation at 95 °C for 10 min followed by 35 cycles of 94 °C for 1 min, annealing for 1 min at 57 °C, and elongation for 1 min at 72 °C, and a final elongation step at 72 °C for 10 min. Expression levels of genes of interest were normalized to that of GAPDH in the same sample.