Qiagenrneasy mini rna cleanup protocol

Liver was taken from mice at 12 months of age, after 8 months exposure to control, rapamycin, or DR diets. Total RNA was isolated from mouse livers using Trizol (Invitrogen, Carlsbad, CA), cleaned using the QiagenRNeasy mini RNA cleanup protocol (Qiagen, Valencia, CA), and quantified using a Nanodrop ND-100. Total RNA was then transcribed to cDNA by using iScriptTMcDNA reverse transcription kits (1708891; BIO-RAD, Hercules, CA). Reverse transcription products were amplified using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and PCR primers (Integrated DNA Technologies, Coralville, IA). RT-PCR was performed using ABI PRISM sequence detection system instrument and software (Applied Biosystems, Inc, Foster City, CA). Ct data, indicating the number of amplification cycles needed to achieve threshold, were available from 6 male and 6 female mice for treatment group. The effect rapamycin or DR diet was estimated as the difference between Ct for the mutant mouse and Ct for sex-matched littermate controls, with a positive value in cases where expression of the mRNA was increased by diet compared to control animals. Because a change of 1 unit for Ct reflects a two-fold change in mRNA abundance, effect sizes in the figures and tables reflect powers of 2. Thus an effect size of 5 indicates an increase in mRNA concentration of 2⁵ = 32-fold.