qPCR Protocol for Gene Expression Analysis

qPCR was performed using StepOne Thermocycler (Applied Biosystems), by Fast ramp speed programme using Luna^® Universal qPCR Master Mix (New England Bio Labs Inc.), and 1 µl of magnum cDNA for the reaction. Thermal cycling conditions were initial denaturation 1 min at 95°C, then 40 cycles of denaturation for 3 s at 95°C and annealing 30 s at 60°C. Each sample was run in triplicate.
Data analysis was conducted using four software: geNorm, NormFinder, BestKeeper and RefFinder (Andersen et al., 2004 (link); Pfaffl et al., 2004 (link); Vandesompele et al., 2002 (link); Xie et al., 2012 ).

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Rodríguez‐Hernández R., Oviedo‐Rondón E.O, & Rondón‐Barragán I.S. (2021). Identification of reliable reference genes for expression studies in the magnum of laying hens housed in cage and cage‐free systems. Veterinary Medicine and Science, 7(5), 1890-1898.

Publication 2021

StepOne Thermocycler Luna® Universal qPCR Master Mix

Cdna

Corresponding Organization : University of Tolima

Other organizations : North Carolina State University

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Variable analysis

independent variables

Fast ramp speed programme using Luna® Universal qPCR Master Mix (New England Bio Labs Inc.)
1 µl of magnum cDNA for the reaction

dependent variables

QPCR results

control variables

StepOne Thermocycler (Applied Biosystems)
Thermal cycling conditions: initial denaturation 1 min at 95°C, then 40 cycles of denaturation for 3 s at 95°C and annealing 30 s at 60°C
Each sample was run in triplicate

controls

No positive or negative controls were explicitly mentioned in the provided information.

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