qRT-PCR Gene Expression Analysis

High quality RNA (DNA free and A_260/280 ~ 2.1) was used as template for the synthesis of first strand of cDNA using SuperScript VILO synthesis kit (Invitrogen). After first strand cDNA synthesis, the product was directly used for qRT-PCR using LighCycer480 Master Mix SYBRGreen in a LightCycler480 Instrument (Roche Life Sciences, USA). For normalization, we used 16S rRNA, as well as gyrB (DNA gyrase subunit B) and/or gapdh (glyceraldehyde-3-phosphate dehydrogenase). The raw data was converted using LC480 Conversion: conversion of raw LC480 data” software (available at http://www.hartfaalcentrum.nl/index.php?main=files&sub=0) and LinregPCR for expression data analysis [58 (link),59 (link)], where the output expression data is displayed in arbitrary fluorescence units (N₀) that represent the starting RNA amount for the test gene in that sample. Statistical significance was determined by performing Student t-test (unpaired samples, one tailed), using GraphPad Prism 6 tool.