Quantitative RT-PCR analysis of a-crystallin expression

Levels of αA-, αBa- and αBb-crystallin expression were measured in wildtype, cloche and cloche sibling embryos using qRT-PCR. Embryos were collected at 4 dpf and chilled on ice before replacing system water with RNAlater (Thermo Fisher) and then stored in a −20 °C freezer until RNA purification. Embryos were stored between 1 h and several days. Approximately 20 embryos were poooled for RNA purification from each genotype for each biological replicate. Three biological replicates were collected from independent fish crosses. Total RNA from each sample was purified using an RNEasy Minikit (QIAGEN) with Qiashreddor and quantified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Purified total RNA (2,000 ng) from each sample was treated with DNaseI (NEB) and 12 μl was used to synthesize cDNA using the Protoscript II First Strand cDNA Synthesis Kit (NEB) with the oligo d(T)23 primer in a total volume of 40 ul. The resulting cDNA sample was calculated to contain the equivalent of 16 ng/μl of original purified RNA.

All further procedures were identical to those described in Posner et al. [5] except that two reference genes were used instead of three (ef1 and rpl13a). In short, three biological replicates for each genotype were amplified in technical triplicate using Luna Universal qPCR Master Mix (NEB) on an Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher). Primer pair design for the two endogenous control genes and three zebrafish α-crystallin genes were previously published [19–21], and we previously validated the efficiency of these primers by standard curve and determined that they produced single amplification products [5]. Reaction conditions were identical to those previously published [5] and identical analysis was done to determine delta Cq values (using recommended MIQE guideline nomenclature [22]), which were then visualized and statistically analyzed using R [23] and R Studio [24].