Fig. 3

Time course analysis of zebrafish PLAG expression by quantitative real-time RT-PCR. Quantitative results are presented as normalized mean (±SD) copies of target genes per 250 ng RNA. Each sample was run in triplicate, together with known dilutions of respective plasmid cDNA ranging from 10⁵ to 10¹ copies, the appropriate non-template controls and mock reverse-transcribed RNA (samples noted w/o RT). For PLAGX real-time PCR runs, melting curve analyses were performed and single specific melting peaks were observed indicating amplification specificity. The absence of cross-reactivity between the different PLAG members was also checked. The normalization factor was calculated using the copy number of 18S in the cDNA sample versus the average 18S copy number in all 12 cDNAs samples. The temporal expression profiles of the PLAG genes were also confirmed using a second set of reverse-transcribed RNAs isolated independently (data not shown).