Total RNA was isolated from approximately 108 cells of C. vulgaris
using TotalRNAExtractor Reagent (Sangon Biotech, China) according
to the manufacturer's instructions. The RNA quantification and purity
were determined by measuring absorption at 260 nm and 280 nm and
by gel electrophoresis. Total RNA (0.7 μg) and the oligo-dT primers
were used for the first strand DNA synthesis using M-MuLV First Strand
cDNA Synthesis Kit (Sangon Biotech, China) according to the manufacturer's
instructions. By using the cDNA as a template, PCR analysis was
performed with primers EGFP-F/EGFP-R and programs mentioned
above. The primers, 18S-F (5′-ACTTCTTAGAGGGACTATTGGCG-3′) and
18S-R (5′-CCTTGTTACGACTTCTCCTTCCT-3′) were used to amplify the
18S rRNA gene fragment (391 bp) as an internal standard.