Abi sybr green pcr master mix

RNA extraction and real-time PCR were conducted as described previously^{36 (link),42 (link)}. Total RNA was extracted from liver and inguinal white adipose tissue (iWAT) using TRIzol reagent following the manufacturer’s protocol (Invitrogen), with the addition of an RNeasy Lipid Tissue Mini Kit (QIAGEN) for iWAT. RNA purity and quantity were determined by spectrophotometry using a NanoDrop (Thermo Scientific). cDNA synthesis was performed with iScript (BioRad) and mRNA was quantified on the ABI 7900 platform using the ABI SYBR Green PCR Master Mix in optical 384-well plates (Applied Biosystems). Primer pairs were designed using the IDT RealTime qPCR Primer Design tool to span an intro-exon boundary (Table S3). Target gene expression was normalized with cyclophilin as the endogenous control.

Total RNA was isolated from control and ppr^A third instar larvae. Five micrograms of total RNA from each sample were reverse transcribed using Random Hexamer Primers and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT—qPCR analysis of the rp49, mitochondrial precursor and mature mitochondrial transcripts were performed in triplicates using 150ng of cDNA per reaction on a 7900HT Real-Time PCR System using ABI SYBR Green PCR Master Mix (Applied Biosystems). An initial activation step for 10 min at 95°C was followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The primer sequences used are provided in S1 Table. Data is presented as mean ± SD. Fold change was calculated as previously described [114 (link)], and statistical significance was determined using a two-tailed Student’s t test (p < 0.05).

Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, USA), according to the manufacturer's instructions. cDNAs were reverse transcribed using a First Strand cDNA Synthesis Kit (Qiagen, Germany). Then, quantitative real‐time PCR (qRT‐PCR) was performed on an ABI SYBR^®Green PCR Master Mix (Applied Biosystems, Foster City, CA). Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) was used as the reference gene. siRNA‐SULT1C2A (GCTCATGCAACCTTCCTCA) and NC‐siRNA‐SULT1C2A (TTCTCCGAACGTGTCACGT) were used to evaluate the expression ratio of SULT1C2A. For qRT‐PCR analysis of rno‐miR‐466c‐5p expression, U6 small nuclear RNA (U6 snRNA) was used as an endogenous control. In addition, melting curves were used to evaluate non‐specific amplification. The primer sequences used in this study are listed in Table 1. Relative expression levels were determined using the 2^−ΔΔCt method.

Total RNA (miRNA and mRNA) was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Subsequently, 1 μg total RNA was reverse transcribed with a specific stem-loop primer for miRNA and with a random primer for mRNA using the AMV First-Strand cDNA Synthesis kit (Fermentas, Pittsburgh, PA, USA). After RT reaction, real-time PCR was performed on a Light Cycler 480 (Roche, Indianapolis, IN, USA) using ABI SYBR-Green PCR Master mix (Applied Biosystems, Bedford, MA, USA). β-actin and small nuclear RNA U6 were used as an internal normalized reference for cDNA and miRNA, respectively. The primers used were as follows: miR-146a forward, 5′-ACACTCCAGCTGGGTGAGAACTGAATTCC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACCCATGG -3′; Smad4 forward, 5′-CTCTAAACCTCAGGCCACATC-3′ and reverse, 5′-CAATACCTCCTCCATCAAAGC-3′; VEGF forward, 5′-ATGAACTTTCTGCTGTCTTGG-3′ and reverse, 5′-TCACCGCCTCGGCTTGTCACA-3′; β-actin forward, 5′-CTCTTCCAGCCTTCCTTCCT-3′ and reverse, 5′-TCATCGTACTCCTGCTTGCT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The RT-qPCR results were analyzed and expressed as the relative miRNA levels of the Ct (cycle threshold) value, which was then calculated to fold change by the value of each control sample set at 1.

RNA extraction and real-time PCR was conducted as described previously^{42 (link)}. Total RNA was extracted from liver, iWAT, and BAT using TRIzol reagent following the manufacturer’s protocol (Invitrogen), with the addition of an RNeasy Lipid Tissue Mini Kit (Qiagen) for the BAT & iWAT. RNA purity and quantity was determined by spectrophotometry using a NanoDrop (Thermo Scientific). cDNA synthesis was performed with iScript (BioRad) and mRNA was quantified on the ABI 7900 platform using the ABI SYBR Green PCR Master Mix in optical 384-well plates (Applied Biosystems). Primer pairs were designed using the IDT RealTime qPCR Primer Design tool with at least one primer spanning an exon-exon boundary. Target gene expression was normalized with cyclophilin as the endogenous control. Primers used are as follows, written 5′ to 3′:
Fas For GGGATCTGGTGAAAGCTGTAG; Rev GTGTTCTCGTTCCAGGATCTG
Scd-1 For CTGTACGGGATCATACTGGTTC; Rev CGTGCCTTGTAAGTTCTGTG
Srebp1 For AGATTGTGGAGCTCAAAGACC; Rev CACTTCGTAGGGTCAGGTTC
Klb For CAGGGATATCTACATCACAGCC; Rev GTAGCCTTTGATTTTGACCTTGTC
Fgf21 For CAAATCCTGGGTGTCAAAGC; Rev CATGGGCTTCAGACTGGTAC
Fgfr1 For AAAGATCTGGTATCCTGTGCC; Rev TCGAGCTAAGCCAAAGTCTG
Fgfr4 For TGTCAAATTCCGCTGTCCAG; Rev ACCACACTTTCCATCACCAG
Cyc For CTTCGAGC TGTTTGCAGACAAAGT; Rev AGATGCCAGGACCTGTATGCT.