HE4 expression levels were determined in each cell line. After collecting cultured cells, total RNA was prepared with an RNeasy Mini kit (Qiagen GmbH) and subsequently reverse transcribed with Superscript IV VILO Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers' protocol. The cDNA was then amplified using the StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with TaqMan Fast Advanced Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) to quantify the mRNA expression of HE4 and ACTB. The optimal thermal cycling conditions for the master mix were as follows: 40 cycles of a two-step PCR (95°C for 1 sec and 60°C for 20 sec) after the initial enzyme activation (50°C for 2 min and 95°C for 2 min). The quantification cycle (Cq) values were determined using StepOne Software v2.0 yielding amplification plots. The ΔΔCq method was then used to calculate relative gene expression, as previously described (39 (link)). The specific TaqMan probes were as follows: HE4, Hs00197437_m1; ACTB, Hs01060665_g1 (Applied Biosystems; Thermo Fisher Scientific, Inc.).
Hs01060665 g1
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Hs01060665_g1 is a laboratory equipment product from Thermo Fisher Scientific. It serves as a core function, but a detailed unbiased description is not available.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Hs02758991 g1, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Taqman universal master mix, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Superscript 4 vilo master mix, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Express one step superscript qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
22 protocols using hs01060665 g1
1
Quantification of HE4 mRNA Expression
2
Quantification of Exosome-associated Genes
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The indicated numbers of LUHMES cells, ACs, and MG were seeded in a plate dish. The next day, 1 or 10 pg/mL IL-6 was added and cells were collected 24 h later. Total RNA including miRNA was purified with an miRNeasy Kit (217084; Qiagen, Venlo, the Netherlands). qRT-PCR assay was then performed as described previously [22 (link)], for which the primer sets were as follows: Hs00233521_m1 for CD9, Hs01041238_g1 for CD63, Hs01002167_m1 for CD81, Hs01075664_m1 for IL-6R, and Hs01060665_g1 for β-actin (Applied Biosystems, Foster City, CA, USA). Values were normalized to those of human β-actin. The comparative cycle time method was used to quantify gene expression. All samples were analyzed in duplicate in each experiment.
3
SARS-CoV-2 Time-Course Infection Assay
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SARS-CoV-2 was handled in Biosafety level 3 (BSL3) facilities throughout. Each cell line was seeded into wells in 48-well plates on the day prior to infection; cells were then infected with SARS-CoV-2 at an MOI of 0.01 for 1 h. After incubation, unbound viruses were removed, and fresh medium was added. At 24, 48 and 72 hpi, total RNA was isolated using a PureLink RNA Mini Kit (Ambion; Thermo Fisher Scientific) and quantified by real-time qRT-PCR analysis using an EXPRESS One-step SuperScript qRT-PCR kit (Invitrogen) and a QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific). The primers and probe sequences (Integrated DNA Technologies, Coralville, IA, USA) targeting the SARS-CoV-2 nucleocapsid gene to detect sub-genomic viral RNA and the RdRp gene to detect viral genomic RNA were described in previous report37 . The primers and probe for ACTB (Hs01060665_g1, Applied Biosystems) transcripts were used as internal controls.
4
Real-Time qPCR Analysis of Cancer Genes
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Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) from 1 x 105 cells of breast cancer cell lines in 24-well plate and reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). We performed real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystems) or THUNDERBIRD SYBR qPCR Mix (TOYOBO) and 7300 Real-Time PCR System (Applied Biosystems) with a set of primers which were purchased from Thermo Fisher Scientific (Waltham, MA, USA, ACTB: Hs01060665_g1, PDL1: Hs00204257-m1, and PDL2: Hs00228839-m1) and were described in Table 1 [22 (link)–24 (link)].
5
Quantitative mTOR and p70S6K Expression in OSCC
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The total RNA of SCC-4 cells, SCC-25 cells, and cancerous and noncancerous tissues obtained from the patients with OSCC was extracted using a TRIzol reagent (Invitrogen; Life Technologies, Carlsbad, CA, USA), and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to synthesize complementary DNA (cDNA). The PCR reaction mixture contained 25 ng of cDNA; 0.5 μL of mTOR gene-expression assay (Hs00234508_m1, Applied Biosystems, Foster City, CA, USA) or β-actin (ACTB) gene-expression assay (Hs01060665_g1, Applied Biosystems, Foster City, CA, USA); and 5 μL of 2× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). qPCR analysis was run in an ABI 7500 Fast Real-Time System (Applied Biosystems, Foster City, CA, USA), and the thermal parameters were 1 cycle of 95 °C for 10 min and 40 cycles of 95 °C for 20 s and 60 °C for 1 min. The threshold cycle (Ct) of the mTOR gene or p70S6K gene was first normalized to the ACTB internal control to obtain the relative threshold cycle (ΔCt), and then the 2−ΔΔCt method was used to calculate the relative expression of target gene.
6
Quantifying CYP3A4 Induction Through RT-PCR
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Total RNA was isolated using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). To measure CYP3A4 induction potencies, the gene expression levels of CYP3A4 were measured by real-time RT-PCR. Real-time RT-PCR was performed with TaqMan Gene Expression Assays (Applied Biosystems). The assay ID for CYP3A4 is Hs00430021_m1 (Applied Biosystems). The cells were treated with 100 nM VD3 (Sigma-Aldrich) for 21 days and 20 μM RIF (FUJIFILM Wako) for 2 days; these agents are known to induce CYP3A4. Controls were treated with dimethyl sulfoxide (final concentration 0.1%; FUJIFILM Wako). Inducer compounds were replaced daily. Relative quantification was performed against a standard curve and the values were normalized against the input determined for the housekeeping gene, GAPDH, and beta-actin. The assay IDs for GAPDH and beta-actin are Hs02758991_g1 and Hs01060665_g1 (all from Applied Biosystems), respectively.
7
Quantifying FGFR2 and ACTB mRNA Expression
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Total RNA was isolated from the cells using an RNeasy mini kit (Qiagen) according to the manufacturer's procedure. Total RNA was reverse‐transcribed using random primers and Superscript II (Invitrogen) and then amplified on a StepOnePlus Real‐Time PCR System with TaqMan Fast Universal PCR Master Mix to quantify expression of FGFR2 and ACTB mRNA. FGFR2 and ACTB TaqMan probes (TaqMan Gene Expression Assay, Hs01552926_m1 and Hs01060665_g1, respectively) were purchased from Applied Biosystems.
8
Transcriptomic Profiling of Colon Cancer
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Snap-frozen fresh samples of colon adenocarcinoma and adjacent normal tissue samples were used for total RNA extraction with Trizol according to the manufacturer's protocol. RNA samples were subjected to quantification and quality control and 50 samples with the highest quality were selected for mRNA and miRNA analysis. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) for both mRNA and miRNA expression detection according to the manufacturer's protocols. Random hexamers were used for mRNA conversion and analysis, whereas specific primers were used for let-7a-5p, miR-544a-3p, and RNU48 analyzes (000377, 002265, and 001006, respectively; Applied Biosystems) according to the manufacturer's protocol.
Predeveloped Taqman® Gene Expression Assay was used for KRAS mRNA expression quantification (Hs00364284_g1, Applied Biosystems), and ACTB (Hs01060665_g1, Applied Biosystems) as an endogenous control. Taqman® MicroRNA assays were used for let-7a-5p and miR-544a-3p (000377 and 002265, respectively; Applied Biosystems) quantification and RNU48 (001006, Applied Biosystems) as an endogenous control. The results of mRNA and miRNA expression were calculated using the comparative Ct method [30] .
Predeveloped Taqman® Gene Expression Assay was used for KRAS mRNA expression quantification (Hs00364284_g1, Applied Biosystems), and ACTB (Hs01060665_g1, Applied Biosystems) as an endogenous control. Taqman® MicroRNA assays were used for let-7a-5p and miR-544a-3p (000377 and 002265, respectively; Applied Biosystems) quantification and RNU48 (001006, Applied Biosystems) as an endogenous control. The results of mRNA and miRNA expression were calculated using the comparative Ct method [30] .
9
Real-time PCR for 14-3-3ζ and USP18 expression
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Real-time PCR (qRT-PCR) assays were performed as before.21 (link) Primers were human 14-3-3ζ primer (Hs00237047_m1, Thermo Fisher Scientific, Waltham, MA) and murine 14-3-3ζ primer (Mm01158417_g1, Thermo Fisher Scientific, Waltham, MA); human USP18 primer (Hs00276441_m1, Thermo Fisher Scientific, Waltham, MA) and murine USP18 primer (Mm01188805_m1, Thermo Fisher Scientific, Waltham, MA); human actin primer (Hs01060665_g1, Thermo Fisher Scientific, Waltham, MA); and murine actin primer (Mm01205647_g1, Thermo Fisher Scientific, Waltham, MA).
10
Heregulin Expression in HER2+ Cancer
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Tissue samples were obtained either before or after trastuzumab-based therapy from patients with HER2-positive breast or gastric cancer who were previously treated by the Kindai University Faculty of Medicine after the study was approved by the Institutional Review Board; the patients provided written informed consent. Total RNA was isolated from paraffin-embedded tissues using the RNeasy Mini Kit and the RNeasy FFPE kit (both from Qiagen, CA, USA) according to the manufacturer's instructions. cDNA was prepared using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, CA, USA), and real-time PCR was conducted to assess the expression of heregulin, along with β-actin as an internal standard, using TaqMan (R) Gene Expression Assays (Applied Biosystems, CA, USA). Primers for heregulin (Hs00247620_m1) and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific (MA, USA). Fluorescence was detected using a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA). The final results were calculated using the ΔΔCt method, normalized to the levels of β-actin as an internal control, and standardized to the median value for each sample. PCR efficiency was evaluated via serial dilution of SK-BR-3 HRG cell cDNA, and the efficiencies were 93% and 91% for β-actin and heregulin, respectively.
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