Quantitative Analysis of Transcript Expression

The RNA from the LCM samples was reverse transcribed to cDNA using the same method previously reported from this laboratory (Kadakkuzha et al., 2013 (link); Raveendra et al., 2018 (link)). 1 μg of RNA was used with Quanta cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer’s instructions and the expression of transcripts were quantified by qRT-PCR using SYBR Green PCR master mix (Applied Biosystems Carlsbad, CA) for detection in ABI 7900 cycler (Applied Biosystems Carlsbad, CA). Quantification of each transcript was normalized to the mouse 18S reference gene following the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link); Kadakkuzha et al., 2013 (link)). One-way ANOVA and Student-Newman test was used to select genes with statistically significant expression levels.