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URL: https://dx.doi.org/DOI:10.17504/protocols.io.mjmc4k6

Authors: Grainne O´Brien; Aleš Tichý, Ales Tichy

Summary: Gene expression - MQRT-PCRReverse transcriptase reactions were performed using High Capacity cDNA Reverse transcription kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer ’ s protocol with 350 ng of total RNA. Multiplex quantitative RT-PCR Real-time PCR was performed using Rotor-Gene Q (Qiagen, Hilden, Germany). All reactions were run in triplicate using PerfeCTa® MultiPlex qPCR SuperMix (Quanta Biosciences, Inc. Gaithersburg, MD, USA) with primer and probe sets for target genes at 300 nM concentration each. 3 ′ 6-Carboxyfluorescein (FAM), 6-Hexachlorofluorescein (HEX), Texas Red and CY5 (all from Eurogentec Ltd, Fawley, Hampshire, UK) were used as fluorochrome reporters for the hydrolysis probes analysed in multiplexed reactions with between 2 and 4 genes per run including the control. Cycling parameters were 2 min at 95°C, then 45 cycles of 10 s at 95°C and 60 s at 60°C. Data were collected and analyzed by Rotor-Gene Q Series Software. Gene target Ct (cycle threshold) values were normalized to a Hypoxanthine-Guanine phosphoribosyltransferase 1 (HPRT1) internal control. Ct values were converted to transcript quantity using standard curves obtained by serial dilution of PCR-amplified DNA fragments of each gene. The linear dynamic range of the standard curves covering six orders of magnitude (serial dilution from 3.2 x 10-4 to 8.2 x 10-10) gave PCR efficiencies between 93% and 103% for each gene with R2 > 0.998. Relative gene expression levels after irradiation were determined relative to unexposed controls.

Affiliations: Cancer Mechanisms and Biomarkers group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health of England, Didcot, United Kingdom, Department of Radiobiology, Faculty of Military Health Sciences, Hradec Králové, University of Defence in Brno, Czech Republic

Version: 1

Publication Date: 2018

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