Total RNA from whole blood was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Foster City, CA, USA) and qRT-PCR experiments were performed using the 7900 Sequence Detection System (Life Technologies). TaqMan pre-developed assay reagents were used to quantify ENOX2 (Hs00197268_m1) and B2M as an internal reference (beta-2 microglobulin, Hs00187842_m1). The Hs00197268_m1 TaqMan expression assay allows the detection of all ENOX2 mRNA splicing variants (Supplementary Figure S2 ). PCR reactions were prepared in duplicate using TaqMan Universal PCR Master Mix (Life Technologies) and ENOX2/B2M ratios were determined using the ∆Ct method.
7900 sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7900 Sequence Detection System is a real-time PCR instrument designed for accurate, sensitive, and reliable gene expression analysis. It provides efficient thermal cycling and data collection capabilities to support a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (7 mentions)
High pure rna isolation kit, by Roche (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (3 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Taqman real time pcr, by Thermo Fisher Scientific (2 mentions)
Tianamp blood dna kit, by Tiangen Biotech (2 mentions)
Imagequant las 4000 camera system, by GE Healthcare (2 mentions)
Taqman master mix, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Abi prism 7900ht system, by Thermo Fisher Scientific (2 mentions)
Taqman microrna rt kit, by Thermo Fisher Scientific (2 mentions)
Taqman r universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
One step sybr green rt pcr reagent kit, by Thermo Fisher Scientific (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
52 protocols using 7900 sequence detection system
1
Quantifying ENOX2 Expression in Whole Blood
2
Quantifying BAP1 and BRCA1 Expression
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Total RNA was extracted from blood mononuclear cells or UT-7 cells using RNAble reagent (Eurobio, Les Ulis, France). Reverse transcription was performed with 2 mg of total RNA using the High-Capacity cDNA reverse transcription kit (Life Technologies). Gene expression was assessed by quantitative real-time polymerase chain reaction (qPCR) relative to the ABL1 control gene using the 7900 Sequence Detection System (Life Technologies). ABL1 mRNA amplifications were performed as recommended by the European Against Cancer collaborative group, and TaqMan predeveloped assay reagents (Life Technologies) were used to quantify BAP1 (HS00184962_m1) and BRCA1 (Hs01556193_m1) transcripts.
3
Quantifying Gene Expression using TRIzol and qPCR
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Total RNA was extracted using TRIzol (Thermo Fisher Scientific K.K., Yokohama, Japan) according to the manufacturer's instructions. The expression of selected genes was determined in triplicate using the 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Each sample was normalized relative to 18S rRNA expression. The following probes were used: ENG, Hs00923996_m1; KRT19, Hs00761767_s1; THY1, Hs00264235_21; and 18S rRNA, Hs99999901_s1 (Applied Biosystems).
4
Quantitative Analysis of FOXM1 and AFP Expression
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Total RNA was extracted using ISOSPIN Cell & Tissue RNA (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Quantitative PCR probes for FOXM1 (Hs1073586_m1) and AFP (Hs00173490_m1) were purchased from Applied Biosystems (Foster City, CA, USA). The expression of selected genes was determined in triplicate using the 7900 Sequence Detection System (Applied Biosystems). Each sample was normalized relative to the expression of a reference gene (18S rRNA). Quantitation of genes expressed in cell lines relative to Huh7 cells was performed using the ΔΔCT method.
5
Genotyping of TP53 Gene Variant
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The TP53 gene rs1042522 C>G was genotyped by using Taqman real-time PCR on a 7900 Sequence Detection System (Applied Biosystems, Foster City, CA), as previously described [51 (link)-53 (link)]. For quality control purposes, eight duplicate positive controls and eight negative controls without DNA were used in each of 384-well plates.
6
Genotyping Four LMO1 Gene SNPs
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About 2 mL of peripheral blood was collected from each subject for genotyping. Four LMO1 gene SNPs (rs110419 A>G, rs4758051 G>A, rs10840002 A>G and rs204938 A>G) identified in a GWAS on neuroblastoma were chosen for genotyping [29 (link)]. Genomic DNA was isolated from peripheral blood leukocytes with a TIANamp Blood DNA Kit (TianGen Biotech, Beijing, China) [38 (link), 40 (link)]. A 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) and Taqman real-time PCR were used to genotype the LMO1 SNPs, as described thoroughly elsewhere [44 (link), 45 (link)]. To obtain convincing results, we performed the genotyping blindly, not knowing whether each subject was a case or control. We also randomly selected 10% of the samples for repeated genotyping, and the genotype concordance was 100%.
7
RNA Isolation and Gene Expression
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Total RNA was extracted using a High Pure RNA Isolation Kit (Roche Diagnostics K.K., Tokyo, Japan) according to the manufacturer’s instructions. Expression of selected genes was determined in triplicate using the 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Each sample was normalized relative to β-actin expression. The following probes were used: ETV4, Hs00383361_g1; MMP1, Hs00899658_m1; and ACTB, Hs999999903_m1.
8
Quantification of Autophagy Markers in Liver
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Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were performed as described previously (27 (link), 28 (link)).
Briefly, total RNA was isolated from the liver tissues using the Rneasy® Mini Kit (QIAGEN, Hilden, Germany). The RNA was reverse-transcribed using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, CA, USA). qRT-PCR was performed by Taqman master mix (Thermo Fisher Scientific, CA, USA) and a 7900 Sequence Detection System (Applied Biosystems, USA). The specific primer information is listed inTable S1
The antibodies used for the immunoblot are LC3B (2775, Cell Signaling Technology, Beverly, MA, USA) and Rubicon (8465, Cell Signaling Technology, Beverly, MA, USA). For signal normalization, anti-GAPDH antibody was used (MAB374, Millipore, Bedford, MA, USA). Membranes were imaged with the ImageQuant LAS 4000 camera system (GE Healthcare). The band intensity was quantified by Image J software.
Briefly, total RNA was isolated from the liver tissues using the Rneasy® Mini Kit (QIAGEN, Hilden, Germany). The RNA was reverse-transcribed using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, CA, USA). qRT-PCR was performed by Taqman master mix (Thermo Fisher Scientific, CA, USA) and a 7900 Sequence Detection System (Applied Biosystems, USA). The specific primer information is listed in
The antibodies used for the immunoblot are LC3B (2775, Cell Signaling Technology, Beverly, MA, USA) and Rubicon (8465, Cell Signaling Technology, Beverly, MA, USA). For signal normalization, anti-GAPDH antibody was used (MAB374, Millipore, Bedford, MA, USA). Membranes were imaged with the ImageQuant LAS 4000 camera system (GE Healthcare). The band intensity was quantified by Image J software.
9
Quantitative RT-PCR and Western Blotting
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RT–qPCR and western blotting were performed as described previously30 (link). Briefly, total RNA was isolated from liver tissues using the RNeasy® Mini Kit (QIAGEN, Hilden, Germany). RNA was reverse-transcribed using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, CA, USA). RT–qPCR was performed using TaqMan Master Mix (Thermo Fisher Scientific, CA, USA) and a 7900 Sequence Detection System (Applied Biosystems, CA, USA). The specific primers used are listed in Supplemental Table 1 . The antibodies used for the immunoblot are listed in Supplemental Table 2 . Membranes were imaged with an Image Quant LAS 4000 camera system (GE Healthcare, IL, USA). The band intensity was quantified by ImageJ software.
10
Quantifying Mitochondrial and Nuclear DNA
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DNA was extracted and purified from the descending thoracic aorta using the Qiagen DNeasy Blood & Tissue Kit and following manufacturer's protocol. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) copy number was measured using a real-time quantitative polymerase chain reaction using an Applied Biosystems 7900 Sequence Detection System. The primer and TaqMan TAMRA probe sequences for the mitochondrial Cytb gene were forward primer (ratCYTB-DNAFWD), 5′-TTCTACCATCCTCCGTGAAATCA-3′; reverse primer (ratCYTB-DNAREV), 5′-GGCCCGGAGCGAGAAG-3′, probe (ratCYTB-DNAPRB), 5′-6FAMCAACCCGCCCACTCGTCCCCTAMRA-3′. The primer and TaqMan TAMRA probe sequences for the nuclear gene MYHC were as follows: forward primer (ratBMYHC-DNAFWD), 5′-AACAGGGCAGACACGGGTT-3′; reverse primer (ratBMYHC-DNAREV), 5′-TGGTCCTCCTTCACAGTCACC-3′, probe (ratBMYHC-DNAPRB), 5′-6FAMTGAGGGCCTGCATGGGCTGTTTACTAMRA-3′.
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