qRT-PCR Analysis of C. elegans Genes

RNA was isolated from ~200 L4 staged (for validation of RNASeq in independent samples) or Day one adult (determination of raga-1 and ins-6 expression in adults) C. elegans samples using method described above. cDNA was synthesized from 30 μg of RNA with SuperScript VILO Master Mix (ThermoFisher Scientific, 11755050) following manufacturer’s instructions. 5 ng cDNA was used as template for each RT-PCR reaction. 2 or three independent biological replicates were used for each genotype/condition and always run in parallel with Taqman control probe to invariant control gene Y45F10D.4 (Ce02467253_g1) (Heintz et al., 2017 (link)), for normalization on a 96 well plate. RT-PCR was performed on the StepOne Plus qPCR Machine (Life Technologies, MA, USA) using Taqman Universal Master Mix II (Life Technologies, 4440040). Taqman probes used to target each gene of interest are as follows: Ce02445578_g1 (C28A5.2), Ce02484227_g1 (F35E12.5 (irg-5)), Ce02421566_m1 (Y39G10AR.6 (ugt-31)), Ce02488119_g1 (K10G4.5), Ce02433249_g1 (ZK84.6 (ins-6)), Ce02489787_g1 (Y116F11B.1 (daf-28)) and Ce02439068_g1 (raga-1). Relative expression levels were calculated using ΔΔCt method.

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Zhang Y., Lanjuin A., Chowdhury S.R., Mistry M., Silva-García C.G., Weir H.J., Lee C.L., Escoubas C.C., Tabakovic E, & Mair W.B. (2019). Neuronal TORC1 modulates longevity via AMPK and cell nonautonomous regulation of mitochondrial dynamics in C. elegans. eLife, 8, e49158.

Publication 2019

SuperScript VILO Master Mix StepOne Plus qPCR Machine Taqman Universal Master Mix II

Adults Biological C elegans Cdna Gene Genotype Raga Rt pcr

Corresponding Organization : Boston University

Other organizations : National Cheng Kung University, Institut de Recherche sur le Cancer et le Vieillissement de Nice, Inserm, Université Côte d'Azur, Centre National de la Recherche Scientifique

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Variable analysis

independent variables

Developmental stage (L4 or Day one adult)
Genotype (unspecified)

dependent variables

Expression levels of raga-1 and ins-6 genes

control variables

Amount of RNA used (30 μg)
Amount of cDNA used (5 ng)
Normalization with Taqman control probe to invariant control gene Y45F10D.4
Parallel processing of biological replicates

positive controls

Taqman control probe to invariant control gene Y45F10D.4 (Ce02467253_g1)

negative controls

Not explicitly mentioned

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