On column rnase free dnase solution

Upon completion of the experiments, we harvested the RNA separately from the cells located on two surfaces of membrane by first scraping the cells on the basal surface using sterilized toothpicks and immediately placing them into 700 μL QIAzol Lysis Reagent (Qiagen, Valencia, CA). Then, the bottom sides of membrane were washed by PBS twice and wiped well by sterile gauze to remove any leftover cells. The cells on the apical surface of the membrane were then harvested in 700 μL QIAzol Lysis Reagent. The total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen). Removal of contaminating genomic DNA was accomplished using an on-column RNase-free DNase solution (Qiagen). One μg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and diluted 1:5 with sterile water.

Total RNA was extracted according to the manufacturer’s instructions using RNeasy Mini Columns (QIAGEN). On-column RNase-free DNase solution (QIAGEN), was applied to degrade contaminating genomic DNA. RNA quantity was assessed with Nanodrop spectrophotometry (ThermoFisher Scientific). Standard reverse transcriptase was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Life Technologies, Foster City, CA). Transcript mRNA levels were determined by qRT-PCR on the ABI Prism 7900HT Real Time System (Applied Biosystems, Carlsbad, CA), using SYBR green (Qiagen). The mouse primer sequences, designed using Primer Express Software Version 3.0 (Applied Biosystems), for the genes measured by SYBR green are provided in Supplementary Table 1. Input RNA was normalized using reference genes (Actb, Gapdh, Hprt, Tuba1a, Tbp) from which the most stable reference gene was determined by the geNorm algorithm.^{(21 )} For each sample, the median cycle threshold (Ct) of each gene (run in triplicate) was normalized to the geometric mean of the median Ct of the most stable reference gene. The delta Ct for each gene was used to calculate the relative mRNA expression changes for each sample. Genes with Ct values > 35 were considered not expressed (NE), as done previously.^{(22 (link))}