RNA samples from HuH-7 cells were extracted from total cell lysate (C+N), nuclear (N) and cytoplasmic (C) fractions (RNeasy mini kit, Qiagen, Germany). Equal amounts of RNA from each fraction were loaded onto 1% formaldehyde agarose gel. Standard Northern blot procedure was performed with an HBc specific probe (Fig. 1 ). 18S and 28S rRNAs were stained with Healthview nucleic acid stain (Genomics, Taiwan). RNA samples were pretreated with amplification grade-DNase I (Sigma-Aldrich, USA) before reverse transcription (High Capacity cDNA Reverse Transcription Kits, Applied Biosystems, USA). Standard protocol of SYBR green qPCR system (Applied Biosystems, 7500) was performed [31] (link). GAPDH mRNA or snRNA U1 were included as an internal control for RT-qPCR.
Sybr green qpcr system
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The SYBR green qPCR system is a real-time quantitative PCR (qPCR) solution that utilizes the SYBR green dye to detect and quantify target DNA sequences. The system provides accurate and sensitive detection of gene expression levels in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Amplification grade dnase 1, by Merck Group (1 mentions)
Healthview nucleic acid stain, by Genomics Plc (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taqtm 2, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7900ht fast rt pcr system, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Ribozol, by Avantor (1 mentions)
4 protocols using sybr green qpcr system
1
RNA Fractionation and Northern Blot
2
Quantitative Analysis of Adipogenic Genes
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Total RNA from mice was isolated using the TRIzol reagent. For RT, 1 µg total RNA was converted into first-strand complementary (c)DNA in 20 µL reactions using TB GreenTM Premix Ex TaqTM II (TaKaRa, USA). The cDNA was analyzed using RT-qPCR on an SYBR Green QPCR system (Applied Biosystems), and the relative abundance of the genes were estimated compared to that of β-tubulin. Specific primers used for mouse Adipoq, Lkb1, Pgc1α, betatrophin, and β-tubulin are described in Supplementary Table S1 .
3
qRT-PCR Analysis of cSCC Biomarkers
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Primary cSCCs and patient-matched normal adjacent skin tissues for quantitative real-time PCR (qRT-PCR) were obtained from consecutive cSCC patients. Total RNA was extracted from cSCC tissues and paired adjacent normal skin tissue using Trizol reagent (Invitrogen, USA). Then, extracted RNAs were reverse-transcribed into cDNA and were then subjected to Taqman qPCR analysis on a 7900HT Fast RT-PCR System (Life Technologies, ThermoFisher, Loughborough, UK). The mRNA levels of S100A9, SPRR2A, and FABP5 were detected by the SYBR Green qPCR system (Life Technologies) using the following primers (Supplementary Table S1 ). GAPDH mRNA level was used as a control. The relative expressions were calculated with 2–ΔΔCT method.
4
Liver, Adipose, and Intestine RNA Expression Analysis
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Total RNA was extracted from livers, epididymal adipose tissue and from intestinal tissues with Ribozol (Amresco, Solon, USA), and complementary DNA was synthesized from 0.5 µg total RNA by reverse transcription using a cDNA SuperMix reverse transcription kit (Quanta, Gaithersburg, USA) with random hexamer primers. Quantitative real-time PCR (qPCR) was conducted in a Step OnePlus Real-Time PCR System using a commercially available SYBR Green qPCR system (Life technologies, Darmstadt, Germany) with specific primers for target genes as described in Supporting Table 2 . Transcripts analysed were cd68, tnfa, il1a, il6, arg1, ym1, col1a1, tgfb1, timp1, mmp9, mmp12, and mmp13 (Supplementary Table 2 ). Relative expression of each gene was quantified by normalization to glycerinaldehyde-3-phosphat-dehydrogenase (GAPDH) mRNA expression.
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