Qpcr primers

Melanoma cells were seeded at a density of 1 × 10⁵ cells ml⁻¹ in 12-well plates 24 h before the experiment. RNA was extracted using TRIzol (Life Technologies) following the manufacturer’s instructions. RNA was treated with DNA-free DNA Removal Kit (Life Technologies), and RNA purity was determined with an ND-1000 Nanodrop (Thermo Fisher Scientific). qPCRs were performed using 100 ng RNA, QuantiTect primer assays and Brilliant III SYBR Green QRT-PCR Kit (Agilent Technologies) in a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as loading control. The following qPCR primers from Qiagen were used: RANBP2 (QT00035378), TPR (QT00046242), OSBPL8 (QT00067102), SUMO1 (QT00014280), NUP50 (QT00081669), ZMPSTE24 (QT00025627) and TOR1AIP1 (QT00070147).

miRs were detected using reverse-transcription quantitative PCR (RT-qPCR) as previously described^{49 (link)}. Oligonucleotides used for reverse transcription and qPCR are shown in Supplementary Table 1. Fibroblast growth factor (FGF) 7 mRNA levels were quantified using qPCR primers (QT00172004, Qiagen, Hilden/Germany), with Quantitect Sybr 2x Master Mix (Qiagen, Hilden, Germany) in 10 µL reactions using the MX3005 qPCR system (Agilent, Santa Clara, California, USA). Transcripts were quantified using standard curve quantification, diluted skin cDNA served as input to generate the standard curve. The geometric mean of transcription factor (TF)IIB and U6 levels were used to normalize for variation in input template. The geometric mean of these two transcripts was unaltered.

RNA was prepared using the Qiagen RNeasy® Mini Kit with the on-column DNase treatment step as per manufacturer’s instructions. A reverse-transcription reaction was carried out using an RT2 (link) First Strand Kit (SA Biosciences, Qiagen Inc., Valencia, CA, USA) to generate cDNA from 1 μg of RNA. A qPCR master mix was prepared and added to the arrays (SA Biosciences) according to manufacturer’s instructions. Real-time PCR detection was performed on an ABI Prism 7500 (ABI Biosystems, Foster City, CA, USA). The threshold cycle (Ct), for each well was calculated using the instrument software. Data analysis was carried out using an online-based analysis template, based on the 2^ΔΔCt method with raw data being normalised to the housekeeping genes on the array plate (http://www.sabiosciences.com). qPCR primers were purchased from Qiagen for the validation of specific genes (TP53 (p53), S100A4, DR3 (TNFRSF25) and c-Myc).

At specified time points after CCI, mice were anesthetized with 4% isoflurane and perfused by cardiac puncture with 30 mL of cold heparinized saline. The brain was then carefully removed and the ipsilateral cortex and hippocampus were dissected and snap frozen for RNA extraction. RNA was isolated with RNeasy Mini kits (QIAGEN, Frederick, MD), following the manufacturer's protocol, and reverse-transcribed to complementary DNA with Superscript III First Strand kits (ThermoFisherScientific, Waltham, MA), according to the manufacturer's protocol. Il17a, RAR-related orphan receptor C (rorc), and interferon-gamma (ifng) genes were measured by real-time RT-PCR (qPCR) using SYBR Green Master mix (2 × ) with ROX (Invitrogen, Carlsbad, CA) on a 7300 Real Time PCR (Applied Biosystems, Waltham, MA) and RT^{2 (link)} qPCR Primers (QIAGEN). Expression fold-change was determined using the delta-delta cycle threshold method and normalized to beta-actin. The following primers were used: NM_010552 RT^{2 (link)} qPCR Primer for mouse il17a; NM_011281 RT^{2 (link)} qPCR Primer for mouse Rorc; NM_008337 RT^{2 (link)} qPCR Primer for mouse Ifng; and NM_007393 RT^{2 (link)} qPCR Primer for mouse Actb.

PA was purchased from Sigma (USA). Antibodies against Rictor, Akt, phospho-Akt S473, IRS-1, phospho-IRS-1 S636/639, and INSR1β were purchased from Cell Signaling Technology (USA). Antibodies against mTOR, phospho-mTOR S2448, S6K1, phospho-S6K1 T389, and GLUT2 were purchased from Santa Cruz Biotechnology (USA). Anti-FFAR1 was purchased from Novus Biologicals (USA). Secondary antibodies used were HRP-linked (GE Healthcare, USA) and Alexa Fluor 594 (Invitrogen, USA). The non-targeting scrambled control siRNA was purchased from Dharmacon (USA) whereas all qPCR primers and the FFAR1 siRNAs were purchased from QIAGEN (Netherlands). Insulin was quantified using Insulin ELISA Kits (Chrystal Chem. INC., USA), and DNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, USA).

Frozen PBMCs were thawed, washed twice with PBS, and RNA was isolated using mirVana miRNA Isolation kit (Ambion, Inc.; Austin, TX). cDNA was generated with 1 μg of RNA using RT² miRNA First Strand Kit (Qiagen; Valencia, CA). Quantitative real time PCR (qRT-PCR) for HBEGF was done using pre-validated qPCR primers (Qiagen) and SYBR Green Fluor qPCR Mastermix (Qiagen) in a CFX real time PCR machine (Bio-Rad-Hercules, CA), and each sample was run in triplicate. Fold-change in expression compared to healthy patients was calculated using delta-delta C_T; mean fold-change is reported.