Protoscript 2 first strand cdna synthesis

mRNA was extracted from X. laevis embryos at stages 2, 9, 10, 12.5, 18, 24, and 30 using 20uL RNAzol (Sigma) per embryo (15–20 embryos per stage) and three biological replicates were used for each stage. cDNA was synthesized from 1μg of mRNA using ProtoScript^® II First Strand cDNA Synthesis (NEB). qPCR was performed using Luna^® Universal qPCR Master Mix (NEB) on a BioRad CFX96 Real Time PCR machine. Each primer set was designed to be specific to each paralog taok1, taok2, and taok3, but able to recognize both the L and S versions of each gene. All normalization was performed using the reference gene slc35b1.L^{61 (link)}. Primers used were: taok1, (5’-CCAACACGAACGAGAGATAC-3’) and (5’CTAGCCTCATGCTTTCAGAG-3’), taok2, (5’- CAGTTCTTACACTGCACAGG-3’) and (5’- CCATTTCTCCACTGTCATCG-3’), taok3, (5’- CAAGGCCCTAAAGAATCACC-3’) and (5’- GCTTGTGAGGCCATCATTTCG-3’), and slc36b1.L, (5’- CGCATTTCCAAACAGGCTCC-3’) and (5’- CAAGAAGTCCCAGAGCTCGC-3’). Cycling conditions were as follows: 95°C x 1 min, then 40 cycles of 95°C x 15s → 60°C x 30s with a plate read after every cycle. Three technical replicates were performed for each of the biological replicates. Efficiency was calculated from the slope of a standard curve generated from qPCR of serially diluted template DNA. Data was normalized to the expression of slc35b1.L using the Livak method (ΔΔC_T).