Lightcycler 480 thermocycler

The gene expression of glial cell-derived neurotrophic factor (GDNF) family receptor alpha 1 (GFRA1) was assessed in the same cohort of samples used in the RNAseq analysis for which sufficient RNA remained (PGES <50 seconds, n = 4, PGES ≥50 seconds, n = 7). PCR primers based on the reported cDNA sequences were designed using the National Center for Biotechnology Information primer design tool.^{28 (link)} The sequences for the forward and reverse primers of GFRA1 were 5′-TCT TCC AGC CGC AGA AGA AC-3′ and 5′-AAC AGT GGG GAC AAA CTG GG-3′, respectively. Total RNA (700 ng) was reverse transcribed into cDNA using oligodT primers. For each quantitative PCR reaction, a mastermix was prepared as follows: 1 µL cDNA, 2.5 µL of 2× SensiFAST SYBR Green Reaction Mix (Bioline Inc, Taunton, MA), and 0.2 µM of both the reverse and forward primers. The PCRs were run on a Roche Lightcycler 480 thermocycler (Roche Applied Science, Basel, Switzerland). Each sample and primer pair were run in triplicates. Data quantification was performed as previously described^{17 (link)} relative to the reference genes, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) and chromosome 1 open reading frame 43 (C1orf43). The normalized ratio was compared between the 2 groups (Mann-Whitney U test); values of p < 0.05 were considered significant.

Total RNA was extracted from mouse tissues with TRIzol Reagent (Life Technologies) in accordance with the manufacturer’s protocol. Reverse-transcription was performed with 1 μg total RNA and the Transcriptor First-Strand Complementary DNA Synthesis Kit (Roche Diagnostics), with random hexamer primers. Quantitative PCR was performed with the Light Cycler 480 Sybr Green I Master kit (Roche) and specific primers (Eurogentec) on a Light Cycler 480 thermocycler (Roche). RNA levels were calculated by the 2^{(-Delta Ct)} method, with 18S as the internal control, relative to RNA levels in control (Axin1^flfl) mice.

Total RNA was extracted with TRIzol reagent (Invitrogen) from ∼3 × 10⁴ purified Purkinje cells at the indicated stages. The corresponding cDNAs were prepared by reverse transcription of 100 ng of RNA using the SuperScript III First-Strand Synthesis System (Invitrogen) with an oligo-dT primer according to the manufacturer's instructions. The resulting cDNAs were used as a template for RT-qPCR using a Light Cycler 480 thermocycler (384 plates; Roche Diagnostics, Meylan, France) with a home-made SYBR Green QPCR master mix (Lutfalla and Uze, 2006 (link)). Thermal cycling parameters were 2 min at 95°C, followed by 45 cycles of 95°C for 10 s, 64°C for 15 s, and 72°C for 25 s. The relative quantification in gene expression was determined using the ΔΔCt method. To normalize expression data, primers for 10 commonly used housekeeping genes were used, and the normalization factor was determined using the geNorm software, as described in Vandesompele et al. (2002) . This led to the selection of the following internal control genes in our assays: glyceraldehyde-3-phosphate dehydrogenase, β-actin, and hypoxanthine phosphoribosyltransferase 1. Sequences of the primers used are listed in Supplemental Table S1.