Quanta sybr green

Total RNA was extracted from pools of six embryos at 4 hr
postfertilization (hpf) or pools of four embryos at 56 hpf using Trizol
(Invitrogen). cDNA was synthesized using SuperScript II reverse transcriptase
(Invitrogen). Quantitative real-time PCR (qPCR) was performed with a Light
Cycler 480 (Roche) using Quanta SYBR Green (Quanta Bioscience). Transcript
levels from each sample were normalized top-actin. Each experiment consisted of
three pools of embryos run in duplicate. The primer pairs used include:
lef1 forward 5′-TCGCCCATGAAAACTCTACTG-3′ and
reverse 5′-TGGACCAAAAGTGACGAGC-3′, dkk1 forward
5′-ACCCACAGGTGAAACAGGAG-3′ and reverse
5′-CAGCATGAAAGCGTTTAGAGG-3′, sp5 forward
5′-GCTTGAGGAACTCGAGGAAG-3’ and reverse 5′-TGTTTTCCGGAGAGGAG
TTC-3′, sp5l forward
5′-CGGACAATTTCCTCCACAAT-3′ and reverse
5′-CTTCCTGCCAAGCCTGACT-3′, and lrrk2 forward
5′-ATGAGGAGGAATGGGATGTG and reverse
5′-CTCCGCCAGGTGATACTCAG-3′.

Total RNA was extracted from pools of eight sphere staged embryos and pools of six shield (6 hpf) and bud (10 hpf) stage embryos using Trizol (Invitrogen) and ZYMO Research Direct-zol RNA MiniPrep. cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen). Quantitative real-time PCR (qPCR) was performed with a Light Cycler 480 (Roche) using QuantaSYBR Green (Quanta Bioscience). Transcript levels from each sample were normalized to β-actin and error bars represent standard error. Each experiment consisted of three pools of embryos run in duplicate with mutants set to 1. Primer pairs used were previously described in (Kok, Oster et al. 2007 (link), Kok, Taibi et al. 2012 (link), Moravec, Samuel et al. 2016 (link)).