Quantification of Transcripts in Mouse Brain

RNA was isolated from the tissues dissected from the adult mouse brain as described earlier and cDNA was generated by reverse transcription as described earlier (Kadakkuzha et al., 2013 (link)). Briefly, 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). All the qPCR amplifications were performed in quadruplicate in a total volume of 10 μl containing 2 μl of H2O, 2 μl of cDNA, 5 μl of 2X Master Mix, 1.0 μl of 10 μM (each) forward and reverse primers. Quantification of each transcript was normalized to the mouse 18S reference gene following the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). Student t-test was used to select genes with statistically significant expression levels where ^*p-value < 0.05, ^**p-value < 0.01, ^***p-value < 0.001. The sequences of primers for the mRNAs and lncRNAs are given in the Table S3.