Primescript reverse transcript

Extraction of total RNA from cells or tissues was performed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription of RNA into cDNA was conducted with PrimeScript Reverse Transcript (Takara, Tokyo, Japan). The RT-PCRs for cDNA amplification were carried out under the following conditions: 95°C for 10 s, 95°C for 30 s, and 40 cycles at 60°C for 30 s. The primers used for RT-PCR were as follows: ALX4, 5′-CAAAGGCAAGAAGCGGCGGAATC-3′ (forward) and 5′-GGTACATTGAGTTGTGCTGTCC-3′ (reverse); β-actin, 5′-AGAAAATCTGGCACCACACC-3′ (forward) and 5′-TAGCACAGCCTGGATAGCAA-3′ (reverse). The relative expression of genes was determined using the comparative CT method (2^−ΔΔCt) (16 (link)). The expression of β-actin was analyzed for normalization.