Quantitative RT-PCR Analysis of C. elegans

Total RNA was isolated using RNAbee (Tel-Test) from approximately 2,000 adult hermaphrodite worms synchronized by bleaching technique at the ages indicated in the corresponding figures. Sample size was determined according to our previous publications^{8 (link),42 (link)}. cDNA was generated using a qScript Flex cDNA synthesis kit (Quantabio). SybrGreen real-time qPCR experiments were performed with a 1:20 dilution of cDNA using the CFC384 Real-Time System (Bio-Rad). Data were analysed with the comparative 2ΔΔC_t method using the geometric mean of cdc-42, pmp-3 and Y45F10D.4 as housekeeping genes^{55 (link)}. qPCR experiments were not blinded. See Supplementary Table 13 for details about the primers used for quantitative RT–PCR.

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Koyuncu S., Loureiro R., Lee H.J., Wagle P., Krueger M, & Vilchez D. (2021). Rewiring of the ubiquitinated proteome determines ageing in C. elegans. Nature, 596(7871), 285-290.

Publication 2021

qScript Flex cDNA synthesis kit CFC384 Real-Time System

Adult Cdna Dilution Hermaphrodite Primers Rt pcr Synthesis Worms

Corresponding Organization :

Other organizations : Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases, University of Cologne

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Variable analysis

independent variables

Age of adult hermaphrodite worms

dependent variables

Gene expression levels

control variables

Total RNA isolation using RNAbee (Tel-Test)
CDNA generation using qScript Flex cDNA synthesis kit (Quantabio)
SybrGreen real-time qPCR experiments using CFC384 Real-Time System (Bio-Rad)
Data analysis using comparative 2ΔΔCt method
Geometric mean of cdc-42, pmp-3 and Y45F10D.4 as housekeeping genes

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