Quanta cdna supermix

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.

RNA was isolated from cell suspensions of rPAEC using Qiagen RNeasy Plus Mini Kit with gDNA filter column (Qiagen, Valecia, CA, USA). RNA was quantified spectrophotometrically (NanoDrop 1000), and equal amounts (1 ug) were loaded with Quanta cDNA Supermix (Quanta Biosciences, Gaithersburg, MD, USA) for cDNA synthesis. cDNA was diluted 1:10 in TE buffer for downstream reactions. Primers for rat-IL6 (Qiagen QuantiTect QT00182896) and rat-ACTB (Qiagen QuantiTect QT00193473) were used in reactions with 100ug cDNA and Quanta PerfectCT a SYBR Green Master Mix. Ninety-six-well PCR plates were run Applied Biosystems AB7300 (ThermoFisher, Waltham, MA, USA), with initial denaturing (95°C, 2 min) followed by 40 cycles of denaturing (95°C, 15s), annealing (55°C, 30s), and extension (72°C, 30s). A melting curve was run to confirm specificity of PCR products. Experimental and reference genes were run on the same plates and relative quantification determined by the ΔC_T method.

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.