Total RNA samples were extracted from cells at specified time-points, RNA was stabilized with Qiagen RNA protect reagent (Qiagen, U.S.A.) and extracted using the PureLink™ RNA Mini kit (Ambion Life Technologies, U.S.A.). Purified RNA was stored at −80°C in nuclease-free water. cDNA was amplified from 100 ng of RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, U.S.A.). qRT-PCR reactions were completed in a final volume of 10 μl (1 μl cDNA, 5 μl TaqMan Fast PCR master mix, 0.5 μl TaqMan probe (both Applied Biosystems, U.S.A.)). Amplification was performed on an Applied Biosystems 7500 Fast Real-Time machine using the following conditions; 50°C 5 min, 95°C 10 min, and 40 cycles of 95°C 15 sec, 60°C 1 min. Real-time analysis was performed in triplicate on RNA from three independent cultures and quantification of 16S expression served as an internal control. The relative expression ratios were calculated using the delta-delta method (PerkinElmer, U.S.A.). Statistical analysis of data was performed by one-way ANOVA, a P-value ≤0.01 was considered to be a significant difference. Primer and probe mixtures were custom designed from Applied Biosystems (Custom TaqMan gene expression Assays (Applied Biosystems, Life Technologies, U.S.A.)) sequences can be viewed in Supplementary Table 2 .
Taqman fast pcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Fast PCR Master Mix is a pre-optimized, ready-to-use solution for fast and efficient real-time PCR amplification. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and buffer, to perform PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Taqman high capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (4 mentions)
Custom taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Rnaprotect reagent, by Qiagen (2 mentions)
7500 fast real time machine, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Realplex4 real time pcr detection system, by Eppendorf (1 mentions)
Hsa mir 103, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mirneasy ffpe kit, by Qiagen (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Abi 7500 fast sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
16 protocols using taqman fast pcr master mix
1
Quantitative gene expression analysis by qRT-PCR
2
Quantifying Gene Expression in Breast Cells
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Total RNA was extracted from BC and noncancerous breast cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For microRNA analysis, RT-qPCR was performed using the TaqMan MicroRNA Reverse Transcription Kit, TaqMan Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the target-specific primers. For mRNA analysis, RT-qPCR was performed using the TaqMan High-Capacity cDNA Reverse Transcription Kit, TaqMan Fast PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the corresponding primers. GAPDH was used as an internal control for normalizing IRAK1 expression levels. RT-qPCR was performed in triplicate on a RealPlex4 real-time PCR detection system from Eppendorf (Hamburg, Germany). The thermocycling conditions for RT-qPCR were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 10 sec. The relative expression levels were calculated using the 2−∆∆Cq method (12 (link)).
3
Quantitative Real-Time PCR Analysis of miRNA
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RNA isolation was performed with the use of a miRNeasy FFPE Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Spectrophotometry (PicoDrop, 260/280 nm) was used in order to assess the quality of the samples. Reverse transcription was performed with the use of the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) and miRNA-specific primers (RT primer), according to the manufacturer’s protocol.
Quantitative Real-time PCR was performed with the use of standard TaqMan® MicroRNA Assays (Applied Biosystems): hsa-miR-103 (Assay ID: 000439), hsa-miR-107 (Assay ID: 000443) and RNU6B (endogenous control, Assay ID: 001093). The 10 μL qPCR reaction mixture included 0.7 μL RT product, 5 μL TaqMan Fast PCR Master Mix (Applied Biosystems) and 1 μL TaqMan miRNA Assay (20×). The reactions were incubated in a 96-well plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. All reactions were run in duplicate (Applied Biosystems 7900HT Fast Real-Time PCR System). We used Detection System 2.3 Software (Applied Biosystems, Waltham, MA, USA) for the quantification of miRNA. Relative expression was calculated according to the Ct method 2−ΔΔCt.
Quantitative Real-time PCR was performed with the use of standard TaqMan® MicroRNA Assays (Applied Biosystems): hsa-miR-103 (Assay ID: 000439), hsa-miR-107 (Assay ID: 000443) and RNU6B (endogenous control, Assay ID: 001093). The 10 μL qPCR reaction mixture included 0.7 μL RT product, 5 μL TaqMan Fast PCR Master Mix (Applied Biosystems) and 1 μL TaqMan miRNA Assay (20×). The reactions were incubated in a 96-well plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. All reactions were run in duplicate (Applied Biosystems 7900HT Fast Real-Time PCR System). We used Detection System 2.3 Software (Applied Biosystems, Waltham, MA, USA) for the quantification of miRNA. Relative expression was calculated according to the Ct method 2−ΔΔCt.
4
Quantitative RNA Expression Analysis
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Total RNA was extracted from CCA tissues and noncancerous cholangiocyte cells using the TRIzol reagent (Invitrogen Corp.) according to the manufacturer’s instructions. For miRNA analysis, quantitative real-time PCR (RT-qPCR) was performed using the TaqMan MicroRNA Reverse Transcription Kit, TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and the corresponding primers. For mRNA analysis, RT-qPCR was performed using the TaqMan High-Capacity cDNA Reverse Transcription Kit, TaqMan Fast PCR Master Mix (Applied Biosystems) and the corresponding primers. RT-qPCRs were performed in triplicate on a RealPlex4 real-time PCR detection system.
5
RNA Extraction and qRT-PCR Analysis of Girdin
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We used TRIzol reagent (Thermo Fisher Scientific, Inc.) for extracting total RNA from GC cells (1 × 107) in line with specific protocols. We adopted TaqMan High-Capacity cDNA Reverse Transcription Kit, TaqMan Fast PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), and related primers for qRT-PCR following specific protocols, with GAPDH being the endogenous reference for normalizing Girdin. The RT-qPCR procedure was conducted as follows: 5 min initial denaturation under 95 °C; then 10 s denaturation under 95 °C, 20 s annealing under 60 °C, and 10 s extension under 72 °C for 40 cycles. Thereafter, the 2−ΔΔCq approach was applied in calculating relative gene expression based on previous description. Sequences of primers used in this study were shown below, for Girdin, 5'-GAGAAGCAGTGGTGGGTTCC-3' (forward) and 5'-GAGCAACACAGATGAACCGC-3' (reverse); for GAPDH 5'-TCCTCTGACTTCAACAGCGACAC-3' (forward) and 5'-CAC CCTGTTGCTGTAGCCAAATTC-3' (reverse).
6
Quantitative Real-Time PCR Analysis
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Total RNA samples were extracted from cells at specified time points, RNA was stabilized with Qiagen RNA protect reagent (Qiagen, USA) and extracted using the PureLink RNA Mini kit (Ambion Life Technologies, USA). Purified RNA was stored at −80 °C in nuclease-free water. cDNA was amplified from 100 ng of RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). qRT-PCR reactions were completed in a final volume of 10 μl (1 μl cDNA, 5 μl TaqMan Fast PCR master mix, 0.5 μl TaqMan probe (both Applied Biosystems, USA)). Amplification was performed on an Applied Biosystems 7500 Fast Real-Time machine using the following conditions; 50 °C 5 min, 95 °C 10 min, and 40 cycles of 95 °C 15 s, 60 °C 1 min. Real-time analysis was performed in triplicate on RNA from three independent cultures and quantification of 16S expression served as an internal control. The relative expression ratios were calculated using the delta-delta method (PerkinElmer, USA). Statistical analysis of data was performed by one-way ANOVA, a P-value ≤0.01 was considered to be a significant difference. Primer and probe mixtures were custom designed from Applied Biosystems (Custom TaqMan gene expression Assays (Applied Biosystems, Life Technologies, USA)) sequences can be viewed in Supplementary Table 2 .
7
Quantitative RT-PCR for Gene Expression
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Total RNA samples were extracted from cells by using RNeasy Plus Micro Kit (Qiagen #74034). RNA integrity was controlled by running a Bioanalizer profile (Agilent) and by measuring their spectral profile at λ = 230, 260 and 280 nm.
300 ng of total RNA was reverse transcribed into cDNA by using the High Capacity cDNA Archive Kit and according to the manufacturer instructions (Applied Biosystems, cat. No. 4368813).
Real-time PCR was performed by using the TaqMan® fast PCR Master Mix (Applied Biosystems, cat no. 4352042) and the ABI prism™ 7900HT (Applied Biosystems) according to the manufacturer instructions.
Relative quantification of gene expression changes was performed by using the standard curve method and normalized to control samples to generate fold changes values. The housekeeping gene used for comparison and normalization of gene expression data was the GAPDH RNA.
Results were presented in fold change as compared to BACE1 expression.
Taqman assays used to assess gene expression: PMEL Mm00498996_m1, BACE1 Mm00478664_m1, BACE2 Mm00517138_m1, PMEL Hs00173854_m1, BACE1 Hs01121195_m1, BACE2 Hs00273238_m1, 18S Hs99999901_s1 Endogenous control, GAPDH Hs02758991_g1 Endogenous control, GAPDH Mm99999915_g1 Endogenous control
300 ng of total RNA was reverse transcribed into cDNA by using the High Capacity cDNA Archive Kit and according to the manufacturer instructions (Applied Biosystems, cat. No. 4368813).
Real-time PCR was performed by using the TaqMan® fast PCR Master Mix (Applied Biosystems, cat no. 4352042) and the ABI prism™ 7900HT (Applied Biosystems) according to the manufacturer instructions.
Relative quantification of gene expression changes was performed by using the standard curve method and normalized to control samples to generate fold changes values. The housekeeping gene used for comparison and normalization of gene expression data was the GAPDH RNA.
Results were presented in fold change as compared to BACE1 expression.
Taqman assays used to assess gene expression: PMEL Mm00498996_m1, BACE1 Mm00478664_m1, BACE2 Mm00517138_m1, PMEL Hs00173854_m1, BACE1 Hs01121195_m1, BACE2 Hs00273238_m1, 18S Hs99999901_s1 Endogenous control, GAPDH Hs02758991_g1 Endogenous control, GAPDH Mm99999915_g1 Endogenous control
8
Cytokine-Induced Differentiation Markers
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Newly confluent cultures were treated with 10 ng/ml IL4, IL6, IL20, or 50 ng/ml OSM. These commonly used concentrations were confirmed to give roughly equivalent phosphorylation of STAT1 in the SIK cultures after one hr of treatment. For measurement of differentiation markers, cells were dissolved in Trizol reagent (Invitrogen) after 3–6 days of treatment, and cDNA was synthesized using high-capacity cDNA Reverse Transcription kit (Applied Biosystems). mRNA levels were measured using real-time PCR with TaqMan Fast PCR Master Mix, TaqMan Gene Expression assays (Applied Biosystems) and ABI 7500 Fast Sequence Detection System. Relative mRNA levels were calculated after normalization of each sample to MAPK1 mRNA. Colony forming efficiency was measured after 10 days of treatment. To this end, 3,000 cells were passaged in 6-cm dishes with a lethally irradiated 3T3 feeder layer, grown until colonies were large enough to see easily (7–14 d), fixed and stained with 0.1% rhodamine in water for counting.
9
Quantifying Osteoclast Regulatory Genes
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To validate our findings from microarray, RNA samples (n = 5–8/group) were converted to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and qPCR was carried out using standard curve methodology. Selection of genes was based on their role in osteoclast differentiation and function and the fold change detected in the data from microarray. Primers (Applied Biosystems) for IL-1α (Rn00566700_m1), IL-1β (Rn00580432_m1), IL-6 (Rn01410330_m1), CCL2 (Rn00580555_m1), Ptgs2 (Rn01483828_m1), LIF (Rn00573491_g1), CD14 (Rn00572656_g1), Csf1 (Rn01522726_m1), RANKL (Rn00589289_m1), OPG (Rn00563499_m1), Acp5 (Rn00569608_m1) and Ctsk (Rn00580723_m1) were used and amplification was performed in duplicate using TaqMan Fast PCR Master mix (Applied Biosystems). Expression levels in samples were normalized to beta-2 microglobulin (Rn00560865_m1) which was expressed steadily between groups.
10
Quantitative Analysis of miRNA and lncRNA Expression
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Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Waltham, CA, USA). For miRNA expression analysis, qRT-PCR was carried out using the TaqMan MicroRNA Reverse Transcription kit and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The relative quanti cation of miR-4684-5p was performed using the 2 -△△Ct method, with U6 used as an internal control. For FAM83H-AS1 and ZBTB38 expression analysis, qRT-PCR was performed using the TaqMan High-Capacity cDNA Reverse Transcription Kit and TaqMan Fast PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The relative quanti cation of FAM83H-AS1 and ZBTB38 was performed using the 2 -△△Ct method, with GAPDH used as an internal control. The reactions were performed independently in triplicate, and the primer sequences are listed in Table 1. The RNA expression levels were analyzed as described previously [13, 21] .
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