RNA analysis

RNA was extracted from homogenized tissue with TRIzol reagent (Ambion Life Technologies) via the manufacturer’s protocol. The quality of the RNA was determined by a NanoDrop 2000 spectrophotometer (ThermoScientific). For each sample, 100 ng of total RNA was reverse transcribed into cDNA with the iScript cDNA synthesis kit (Bio-Rad Laboratories). RT-qPCR was performed for nAChR subunits, transthyretin (TTR), mir-204 and the housekeeping genes, β-actin (ACTB) or U6 (RNU6). TaqMan Universal Master Mix II with real-time PCR gene expression assays for CHRNA3, CHRNA5, CHRNA7, CHRNA4, CHRNA6, CHRNA9, CHRNA10, CHRNB4, CHRNB2, CHRNB3, TTR, mir-204, and ACTB (control) or microRNA assay for miR-204 and RNU6 (control) were used according to manufactures parameters (Applied Biosystems). Samples were tested in duplicate or triplicate (depending on quantity of RNA available per dissection) and quantified with a CFX96 RT-qPCR system (Bio-Rad). Samples with Ct values >35 cycles were considered outside of the range of inclusion as predetermined criteria, and thus, these samples were determined to exhibit no RNA expression in the tissue of interest. Normalized gene expression (2^ΔCt) was calculated with the equation 2^^{(β-actin Ct – target mRNA Ct)}. For nAChR subunit genes, normalized values were multiplied by 10,000 to represent data as whole numbers, and for the miRNA gene expression assay, normalized values were multiplied by 1000. For the microRNA assay, normalized mi-204 expression (2^ΔCt) was calculated with the equation 2^^{(U6 Ct – target miRNA Ct)}. After samples were processed, group assignment was revealed to permit comparisons of the data.