RNA Extraction and qPCR Analysis

For human cell samples, total RNA was extracted using RNAbee (Tel-Test Inc.). cDNA was generated using qScript Flex cDNA synthesis kit (Quantabio). SybrGreen real-time qPCR experiments were performed with a 1:20 dilution of cDNA using a CFC384 Real-Time System (Bio-Rad) following the manufacturer's instructions. Data were analysed with the comparative 2ΔΔC_t method using the geometric mean of ACTB and GAPDH as housekeeping genes. For C. elegans samples, total RNA was isolated from synchronized populations of ∼2,000 adults using QIAzol lysis reagent (Qiagen). Data were analysed with the comparative 2ΔΔC_t method using the geometric mean of cdc-42 and pmp-3 as endogenous control75 (link). See Supplementary Tables 4 and 5 for details about the primers used for this assay.

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Noormohammadi A., Khodakarami A., Gutierrez-Garcia R., Lee H.J., Koyuncu S., König T., Schindler C., Saez I., Fatima A., Dieterich C, & Vilchez D. (2016). Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan. Nature Communications, 7, 13649.

Publication 2016

RNAbee qScript Flex cDNA synthesis kit CFC384 Real-Time System QIAzol lysis reagent

Adults Assay C elegans Cdna Cell Dilution Gapdh Housekeeping genes Human Populations Primers Synthesis

Corresponding Organization :

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

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression of target genes

control variables

RNA extraction method (RNAbee for human samples, QIAzol for C. elegans samples)
CDNA synthesis kit (qScript Flex)
QPCR detection method (SybrGreen)
Housekeeping genes for data normalization (ACTB and GAPDH for human samples, cdc-42 and pmp-3 for C. elegans samples)

controls

Positive control: None mentioned
Negative control: None mentioned

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