Quantitative real-time RT-PCR was performed in triplicate with an Applied Biosystems Prism 7,500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacture's protocol (Applied Biosystems Inc., Foster City, California, USA). TaqMan probes and primers were purchased from Applied Biosystems Inc. Levels of RNA expression were determined using the 7,500 Fast System SDS software package (version 1.3.1; Applied Biosystems Inc.) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a control for normalization. Primers used here were as follows: GAPDH for: 5′-TGACTTCAACAGCGACAC- -CCA-3′,GAPDH, reverse:5′-CACCCTGTTGCTGTAGCCAAA−3′, DGKZ for: 5′-AGCAAG–CAAGAAGAAGAAGAGG-3′, and DGKZ reverse:5′-GGATTGAGATACCAGAGGAAAGAC–3′. The relative DGKZ expression was normalized to GAPDH, and data analysis was conducted using the comparative CT method.
Prism 7500 fast sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Prism 7500 Fast Sequence Detection System is a real-time PCR instrument designed for high-throughput, reliable, and sensitive nucleic acid detection and quantification. It is capable of performing fast PCR protocols and supports a wide range of fluorescent dye chemistries and sample formats.
Automatically generated - may contain errors
Lab products found in correlation
Taqman universal pcr master mix, by Thermo Fisher Scientific (12 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
7500 fast system sds software version 1, by Thermo Fisher Scientific (5 mentions)
Taqman probes and primers, by Thermo Fisher Scientific (3 mentions)
Taqman mirna reverse transcript kit, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Iscript reverse transcription supermix, by Bio-Rad (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Tgfb1, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna, by Thermo Fisher Scientific (1 mentions)
Iscript synthesis kit, by Bio-Rad (1 mentions)
20 protocols using prism 7500 fast sequence detection system
1
Quantitative Analysis of DGKZ Expression
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden Germany) following the manufacturer's instruction. Genomic DNA was removed from RNA extracts by on-column DNase digestion. Synthesis of cDNA was performed using 500 ng of total RNA and iScript reverse transcription Supermix in 10 μl reverse transcription (RT) reaction (Bio-Rad, Hercules, CA), following the manufacturer's instructions. Comparative real-time qPCR was performed using TaqMan Universal PCR Master Mix in triplicate in Applied Biosystems Prism 7500 Fast Sequence Detection System, according to the manufacturer's instructions. TaqMan probes and primers were purchased from Applied Biosystems: Human COL1A1 (Hs00164004_m1), SERPINE1 (Hs00167155_m1), CCN2 (CTGF) (Hs00170014_m1), MMP2 (Hs01548727_m1), ACP5 (Hs00356261_m1), ATP6V0D2 (Hs01084784_m1), DCSTAMP (Hs00229255_m1), and TGFB1 (Hs00998133_m1). Human GAPDH (Hs02786624_g1) and TBP (Hs00427620_m1) were used as endogenous controls for normalization of real-time qPCR in osteoclast RNA and human GAPDH (Hs02758991_g1) was used as an endogenous control for normalization of gene expression in SMAD3-transfected cells. Relative expression was calculated using the comparative ΔΔCt method.
3
Quantitative Analysis of miRNA and mRNA Expression
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TRIzol reagent (Invitrogen) was used to extract total RNA. Reverse transcription was conducted using TaqMan miRNA Reverse Transcript Kit (Applied Biosystems, USA). The PCR was performed on the Prism 7500 FAST Sequence Detection System of Applied Biosystems. Primers used in this study include miR-490-3p (forward: 5′-CGTGGATCCTTCTTCAACCAACGGTGGTG-3′, reverse: 5′-CCAGAATTCAAAGCAGGAAGAGTAAGACTTCC-3′), TNKS2 (forward: 5′- CGCGGATCCTGAAGGTATGGTCGATG-3′, reverse: 5′- CGCGAATTCAATTTAGTACAGACAACCC-3′), PCBP1 (forward: 5′-CAGTGCGGCTCCCTGATTG-3′, reverse: 5′- CCTCTGGAGAGCTGGAGTCAATTC-3′), RASAL2 (forward: 5′-TCCCTCGTGTTCTTGCTGAT-3′, reverse: 5′- GTCTGTGTTGTCCTGGCTTG-3′), and GAPDH (forward: 5′-AACGGATTT GGTCGTATTG-3′, reverse: 5′-GGAA GATGGTGATGGGATT-3′).
4
Quantitative Real-Time RT-PCR Analysis
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Quantitative real-time RT–PCR was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacture's protocol (Applied Biosystems). The TaqMan probes and primers were purchased from Applied Biosystems. RNU48 and GAPDH were used as endogenous control. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems). The mRNA and miRNA expression levels were determined using the 2–ΔCt or 2–ΔΔCt method.
5
Quantitative Analysis of MLH1 Expression
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Extracted RNA was reverse-transcribed into complementary DNA (cDNA) using iScript cDNA Synthesis kit (Bio-Rad) and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR analysis was performed with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan Universal PCR master mix according to the manufacturer's protocol (Applied Biosystems). Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems). PCR parameters for cycling were as follows: 95°C for 20 seconds, then 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds. All reactions were done in a 10 μL reaction volume in triplicate. The data were analyzed using the delta-delta Ct method to calculate the fold-change. TaqMan probes and primers for MLH1 (assay ID: Hs00179866_m1) and GAPDH (assay ID: Hs02758991_g1) were obtained from Applied Biosystems. GAPDH was used as internal control.
6
Quantitative Analysis of Osteoblast Gene Expression
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Total RNA was extracted from osteoblasts using the RNeasy Mini kit (Qiagen) following the manufacturer’s instruction. Genomic DNA was removed from RNA preps by on-column DNase digestion. Synthesis of cDNA was performed using 500 ng of total RNA and iScript reverse transcription Supermix (Bio-Rad), following the manufacturer’s instructions. Comparative real-time qPCR was performed in TaqMan Universal PCR Master Mix in triplicate using Applied Biosystems Prism 7500 Fast Sequence Detection System, according to the manufacturer’s protocol. TaqMan probes and primers were purchased from Applied Biosystems: Human COL1A1 (Hs00164004_m1), COL1A2 (Hs01028956_m1), CDKN2B (Hs00793225_m1), SERPINE1 (Hs00167155_m1), ALPL (Hs01029144_m1), SP7 (Hs01866874_s1), CDH11 (Hs00901479_m1), MMP13 (Hs00942584_m1), and SPP1 (Hs00959010_m1). Mouse Ctgf (Mm01192933_g1), Serpine1 (Mm00435858_m1), and Vegfa (Mm00437306_m1). Human GAPDH (Hs02786624_g1), human TBP (Hs00427620_m1), mouse Gapdh (Mm99999915_g1), and mouse Tbp (Mm00446971_m1) were used as endogenous controls for normalization of real-time qPCR. Relative expression was calculated using the comparative ΔΔCt method.
7
Osteoblast gene expression analysis
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Total RNA was extracted from osteoblasts using the RNeasy mini kit (Qiagen) following the manufacturer’s instruction. RNA concentration was measured by the NanoDrop spectrophotometer. Synthesis of cDNA was performed using 1 µg of total RNA and the High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Comparative real-time PCR was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan universal PCR master mix, according to the manufacture’s protocol (Applied Biosystems Inc., Foster City, CA) (Fig. 5a, f, g ). The TaqMan probes and primers were purchased from Applied Biosystems: Human RUNX2 (Hs01047973_m1), COL1A1 (Hs00164004_m1), ALPL (Hs01029144_m1), RANKL (TNFSF11, Hs00243522_m1), OPG (TNFRSF11B, Hs00900358_m1). Human GAPDH (Hs02786624_g1) and TBP (Hs00427620_m1) were used as endogenous controls for normalization. Levels of MAP2K1, RUNX2, ALPL and COL1A1, RANKL (TNFSF11), and OPG (TNFRSF11B) transcripts were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems). Relative expression was calculated using the comparative ∆∆Ct method.
8
Quantitative RNA Analysis Protocol
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Total RNA was isolated using TRIzol Reagent (Invitrogen) following the manufacture's protocol. qRT-PCR was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System, using TaqMan universal PCR master mix according to the manufacture's protocol (Applied Biosystems). Gene expression levels relative to 18S rRNA were calculated by gthe 2−ΔΔCT method. The primer sequences used in RT-PCR are listed in Table S2 .
9
Quantitative Gene Expression Analysis
10
Comprehensive RNA Extraction and qPCR Analysis
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Total RNA from cells and fresh-frozen tissues were extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. For FFPE samples, RNA was extracted from three 5-μM-thick LUAD FFPE specimens with the RNeasy FFPE Kit (Qiagen, Germany). For qRT-PCR, cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, China). The reaction was carried out for 15 min at 37 °C, 5 min at 85 °C, and then 4 °C until further use.
For qPCR, the expression of genes was measured with the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA) in triplicate using the Applied Biosystems Prism 7500 Fast Sequence Detection System (USA). GAPDH, ACTB, and snRNA U6 were used as the internal controls. The relative level of each target RNA was calculated using the 2△△Ct method and normalized to ACTB. All primer sequence information is listed in Additional file2 : Table S8, and all reagent and kit information is listed in Additional file 2 : Table S9.
For qPCR, the expression of genes was measured with the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA) in triplicate using the Applied Biosystems Prism 7500 Fast Sequence Detection System (USA). GAPDH, ACTB, and snRNA U6 were used as the internal controls. The relative level of each target RNA was calculated using the 2△△Ct method and normalized to ACTB. All primer sequence information is listed in Additional file
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