Improm 2 reverse transcriptase kit

The quantitative real time PCR (qRT-PCR) technique was performed as described previously.^{35 (link), 36 (link)} Briefly, total RNA was isolated from 300 mg of the liver from Con and experimental mice using TRIzol REAGENT (Sigma-Aldrich, Milan, Italy), according to the manufacturer's instructions. RNA (50 ng) was retrotranscribed using the ImProm-II Reverse Transcriptase Kit (Promega, Milan, Italy) to obtain cDNA, which was amplified using the StepOnePlus Real-Time PCR System (Life Technologies, Monza, Italy). Quantitative Real-Time PCR (qRT-PCR) was performed using GoTaq qPCR Master Mix (A6001; Promega). The mRNA levels were normalized to the levels obtained for hypoxanthine phosphoribosyltransferase 1, for β-glucuronidase, and for glyceraldehyde-3-phosphate dehydrogenase. The cDNA was amplified using the primers indicated in Supplementary Table S1 online and the Rotor-gene 6000 Real-Time PCR Machine (Qiagen GmbH, Hilden, Germany). Changes in the transcript level were calculated using the 2^–ΔΔCT method.^{37 (link)}

qRT–PCR was performed as described previously (Seo et al. 2013 (link)). Synchronized worms treated with sbp-1 RNAi or mdt-15 RNAi from eggs with or without 2% glucose treatment were harvested at young adult stage (day 1) with M9 buffer by washing three times. Synchronized worms were cultured on control RNAi with or without 2% glucose until L4 stage and then transferred onto pod-2 RNAi or fasn-1 RNAi plates. The pod-2 RNAi-treated or fasn-1 RNAi-treated worms were harvested after 24 h with M9 buffer by washing three times. RNA was isolated using RNAiso plus (Takara) and reverse-transcribed to cDNA using ImProm-II reverse transcriptase kit (Promega). Random primers (9-mers; Cosmogenetech) were used for reverse transcription except for the measurement of sbp-1 mRNA (Supplemental Fig. S6C), which was reverse-transcribed with oligo(dT) 15 primer (Promega). Quantitative real-time PCR was performed by using the StepOne real-time PCR system (Applied Biosystems), and relative quantity was analyzed by using comparative Ct methods described in the manufacturer's manual. The average of two technical repeats was used for each biological data set. mRNA levels of ama-1, which encodes an RNA polymerase II large subunit, were used for normalization. See the Supplemental Material for the sequence information of the primers used for the assays.