Cfx384 real time system

DEseq2 [26 (link)] was used to identify DE genes and lincRNAs between high- and low-FE pigs. Annotated protein-coding genes and lincRNAs showing |log2FoldChange| (|log2FC|) ≥ 1 and p-value < 0.05 were considered to be DE.
The relative expression levels of DE genes in adipose tissues were quantified through a real-time quantitative PCR (RT-qPCR) detecting system. Total RNA was extracted for RNA sequencing as described. Complementary DNA (cDNA) fragments were obtained via reverse transcription by using a RevertAid First Strand cDNA Synthesis Kit (K1621, Thermo Scientific, Waltham, MA, USA). Oligonucleotide primers for three DE genes and three lincRNAs were designed with Primer7 software. Primer sequences are listed in Supplementary Table S1. qPCR was conducted with SYBR Green PCR MasterMix (Toyobo Co., Ltd., Osaka, Osaka Prefecture, Japan) on a Bio-Rad CFX384 Real-Time System (BioRad Laboratories, Inc., Hercules, CA, USA) in accordance with the manufacturer’s instruction. The 2^−ΔΔCt method was applied to normalize the relative expression levels of DE genes and lincRNAs to those of the ACTB gene, which is stably expressed in adipose tissues (Figure S1) [27 (link)]. The Student’s t-test was used to analyze the differential expression of genes between high- and low-FE pigs.

RNA from liver biopsies was extracted using the RNeasy Mini Kit (QIAGEN, Frederick, MD, USA). RNA concentration was assessed spectrophotometrically. mRNA expression of IL-8, IL-18, IL-33, TNF-α, IFNG, COL1A2, ACTA2, KRT7 and KRT19 were analyzed in triplicate by quantitative real-time polymerase chain reaction (qPCR) using Custom RT Profiler PCR Array (CAPH12366A) (QIAGEN SABiosciences, Frederick, MD, USA) on BIO-RAD CFX384 Real-Time System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. ACTA2 (marker of myofibroblast activation) and COL1A2 (marker of collagen production) were studied as surrogates for active liver fibrogenesis, and KRT7 and KRT19 as cholangiocyte markers, while HPRT1, GAPDH, ACTB and B2M were used as housekeeping genes. Quantification of target gene mRNA expression was performed using the ΔΔCt method and expressed after normalization to housekeeping genes and relative to normal control subjects.

Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Piscataway, NJ, USA) was used for WGA. The lysis (400 mM KOH, 10 mM EDTA, 100 mM DTT), neutralization (0.4 ml of 1 M HCl and 0.6 ml of 1 M Tris·HCL, pH 7.5), and pushing buffers (1X Tris·EDTA, 0.2% Tween-20) were introduced to the dedicated inlets on the device. The amplification reaction was conducted on an AmpliSpeed slide cycler (Advalytix AG, Munich, Germany) for 2 h set at 34°C. After amplification, the samples were collected from the individual amplification units and transferred to a PCR plate followed by an inactivation step at 65°C for 10 min. For re-amplification of the on-chip amplified samples, 17 µl of the WGA mixture was added to 1 µl of on-chip amplified sample based on the manufacturer’s protocol and the reaction was carried out at 30°C on a Bio-Rad CFX 384 Real-Time System (Bio-Rad, Hercules, CA, USA,) for 70 min. This was followed by an inactivation step at 65°C for 10 min.

qRT-PCR was used to verify the RNA-Seq data. LncRNAs were chosen based on expression level and biological significance. Sixteen μL of total RNA was used to synthesize the first strand of cDNA using PrimeScript™ RT Master Mix (Catalog No. RR036A, Takara, Japan). Real-time PCR was performed using TB Green (Catalog No. RR420A, Takara, Japan) in 96-well plates using the Biorad CFX384 Real-Time System (Bio-Rad, CA). The relative levels of target exosome-packaged lncRNAs were normalized against a synthesized exogenous reference λ polyA+ RNA (Catalog No. 3789, Takara, Japan).

Total RNA was isolated from cell lysates using NucleoZol (TaKaRa Bio) according to manufacturer’s instructions, and 1 μg of total RNA was suspended in 10 μl of RNase-free water. RT-qPCR was performed with 1 μl of 1:10 dilution of the RNA using Luna Universal One-Step RT-qPCR kit (New England BioLabs), with a total reaction volume of 10 μl in a Bio-Rad CFX384 Real-Time System (Bio-Rad). Primers used for Ago2 [90 (link)], Dicer [72 (link)], GPRC5A [62 (link)], SUMO3 [91 (link)] and SUMO1, SUMO2, and β-actin [23 (link)] transcripts are as previously described. The relative mRNA expression was derived from 2^-ΔΔCT by use of the comparative threshold cycle (CT) method. The abundance of mRNA in each sample was normalized to the amount of actin mRNA.

Total RNA from cells exposed to extracts for 48 h was extracted using TRIzol reagent (Invitrogen) and quantified using a nano-drop followed by Reverse Transcriptase PCR using random primers per manufacturer’s recommendations. The 48 h time interval has been used as standard in the tobacco field to assess the effect of tobacco exposure in diverse cellular mechanisms including cisplatin resistance^{35 (link)}. To determine the levels of mRNA expression, the first-strand cDNA was amplified using KAPA SYBR FAST Universal (KAPA Biosystems) on a BioRad CFX384 Real-Time System, BioRad, California, USA. The sequences of primers used are listed in Supplementary Table S2. β-actin was used as an endogenous control. Two to 3 experiments with 6 replicates each.