Total RNA was isolated from the whole embryos (24 hpf stage) using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. Poly A RNA was isolated using oligo(dT)25 beads (Dynal Biotech ASA, Oslo, Norway) according to the manufacturer’s instruction.
Description
SAGE analysis was carried out as described (Velculescu et al., 1995) but was modified slightly according to Lee et al (Lee et al., 2007). We performed a low-cycle PCR (14 cycles) using the GeneAmp PCR System 2400 (Applied Biosystem, Foster City, CA) to amplify 3’cDNA in order to generate a sufficient amount of templates for SAGE analysis. The sense primer used was a SAGE primer 1 (5’-GGA TTT GCT GGT GCA GTA CA-3’) or SAGE primer 2 (5’-CTG CTC GAA TTC AAG CTT CT-3’); the antisense primer used was 5’-biotin- ACT ATC TAG AGC GGC CGC TT-3’, which was located in the 3’ end of all cDNAs generated from the anchored oligo(dT) primers.
