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Abi 7900ht sequencing detection system

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The ABI 7900HT Sequencing Detection System is a real-time PCR instrument designed for high-throughput gene expression analysis and SNP genotyping. It features a 384-well format, a flexible optical system, and advanced software for data analysis.

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Lab products found in correlation

High capacity cdna archive kit, by Thermo Fisher Scientific (9 mentions) Taqman gene expression master mix, by Thermo Fisher Scientific (8 mentions) Rneasy mini kit, by Qiagen (6 mentions) Rneasy rna purification kit, by Qiagen (6 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions) Cd206, by Thermo Fisher Scientific (5 mentions) Tnf α, by Thermo Fisher Scientific (5 mentions) Nanodrop 1000, by Thermo Fisher Scientific (3 mentions) 18s rrna, by Thermo Fisher Scientific (3 mentions) Arginase 1, by Thermo Fisher Scientific (3 mentions) High capacity rna to cdna kit, by Thermo Fisher Scientific (3 mentions) Iscript cdna synthesis kit, by Bio-Rad (2 mentions) Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions) Il 1β, by Thermo Fisher Scientific (2 mentions) Il 1ra, by Thermo Fisher Scientific (2 mentions) Taqman primer, by Thermo Fisher Scientific (1 mentions) Thermocycler, by Eppendorf (1 mentions) Mcp 1, by Thermo Fisher Scientific (1 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions)

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ABI 7900HT (358 citations) 7900HT system (158 citations) ABI 7900HT system (132 citations)

20 protocols using abi 7900ht sequencing detection system

1

Quantifying Gene Expression in Cells

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Total RNA was extracted using Qiagen’s RNeasy Mini Kit (Valencia, CA). The amount of the total RNA was measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific). Total RNA (100 ng/sample) was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems (Foster City, CA). qRT-PCR was performed using TaqMan Gene Expression Master Mix and primer probes for 18s, iNOS, TNF-α, CD206, Arg1, and ABI 7900HT Sequencing Detection System (all from Applied Biosystems). Housekeeping gene 18s was used as the internal control. Relative gene expression level to gene 18s was quantified with the comparative Ct method.
Gibon E., Loi F., Córdova L.A., Pajarinen J., Lin T., Lu L., Nabeshima A., Yao Z, & Goodman S.B. (2016). Aging Affects Bone Marrow Macrophage Polarization: Relevance to Bone Healing. Regenerative engineering and translational medicine, 2(2), 98-104.
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2

Quantitative RT-PCR Analysis of Murine Macrophages

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Cellular ribonucleic acid (RNA) was extracted from the lysates of murine macrophages using RNeasy Mini kit, and the amount of purified RNA was measured with a NanoDrop 1000 spectrophotometer (Thermo scientific, Waltham, MA). An equal amount of total RNA from each sample was directed into reverse transcription, and the complementary DNA (cDNA) was synthesized in a thermocycler using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). A reaction mix was prepared containing sample cDNA, a specific TaqMan primer-probe and TaqMan Gene Expression Master Mix (Applied Biosystems). The quantitative real-time PCR was performed with ABI 7900HT Sequencing Detection System (Applied Biosystems) for TNF, IL-1β, IL-6, IL-1Ra, iNOS, CCL2, CXCL9, MRC1, and TLR4 (all primer-probes from Applied Biosystems). The comparative Ct method was used to obtain the results with 18S ribosomal RNA serving as an internal control.
Jämsen E., Pajarinen J., Lin T.H., Lo C.W., Nabeshima A., Lu L., Nathan K., Eklund K.K., Yao Z, & Goodman S.B. (2019). Effect of Aging on the Macrophage Response to Titanium Particles. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 38(2), 405-416.
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3

RNA Extraction and qRT-PCR Analysis

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Total RNA was extracted using RNeasy Mini kit following manufacturer’s instructions. The quality and the amount of the total RNA were measured with a NanoDrop 1000 spectrophotometer (Thermo scientific). Complementary DNA (cDNA) was synthesized using an equal amount of total RNA from each sample and a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). For quantitative real-time PCR, a reaction mix was prepared containing sample cDNA, one of 18S, TNF-α, IL-1Ra, CD206, Arg1, IRF4, IRF5, iNOS or CXCL9 TaqMan primer-probes and TaqMan Gene Expression Master Mix (all from Applied Biosystems). qRT-PCR was performed with ABI 7900HT Sequencing Detection System (Applied Biosystems) using 18S rRNA as an internal control. The final results were obtained by use of the comparative Ct method [25 ].
Pajarinen J., Tamaki Y., Antonios J.K., Lin T.H., Sato T., Yao Z., Takagi M., Konttinen Y.T, & Goodman S.B. (2014). Modulation of Mouse Macrophage Polarization in vitro Using IL-4 Delivery by Osmotic Pumps. Journal of biomedical materials research. Part A, 103(4), 1339-1345.
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4

Spinal Cord mRNA Expression Analysis

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At 4 weeks after fracture, the rats were killed by CO2 inhalation and the ipsilateral spinal cord (L4,5 lumbar enlargement) was collected after behavioral testing and frozen immediately on dry ice. In mice, the spinal cord was collected at 3 weeks after fracture. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands), and the purity and concentration were determined spectrophotometrically. Next, cDNA was synthesized from 1 μg RNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Real-time polymerase chain reactions (PCRs) were conducted using the SYBR Green PCR master mix (Applied Biosystems, Waltham, MA) and performed on an ABI 7900HT sequencing detection system (Applied Biosystems). To validate the primer sets used, we performed dissociation curves to document single-product formation, and agarose gel analysis was conducted to confirm the size (Table 1). The data from real-time PCR experiments were analyzed as described in the manual for the ABI prism 7000 real-time systems. All results were confirmed by repeating the experiment 3 times.
Shi X., Guo T.Z., Wei T., Li W.W., Clark D.J, & Kingery W.S. (2015). Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to postfracture nociceptive sensitization. Pain, 156(10), 1852-1863.
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5

Quantitative Analysis of Gene Expression

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Total RNA was extracted using an RNeasy Mini kit following the manufacturer’s instructions. The quality and the amount of the total RNA were measured with a NanoDrop 1000 spectrophotometer (Thermo scientific). Complementary DNA (cDNA) was synthesized from an equal amount of total RNA from each sample using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) and a thermocycler (Eppendorf, Hamburg, Germany). For quantitative real-time PCR, a reaction mix containing sample cDNA, one of 18S, TNF-α, iNOS, IRF5, CD206, Arg1 or IRF4 TaqMan primer-probes and TaqMan Gene Expression Master Mix (all from Applied Biosystems) was prepared. qRT-PCR was performed with ABI 7900HT Sequencing Detection System (Applied Biosystems) using 18S rRNA as an internal control. The results were obtained by use of the comparative Ct method [9 (link)].
Pajarinen J., Lin T.H., Sato T., Loi F., Yao Z., Konttinen Y.T, & Goodman S.B. (2015). Establishment of Green Fluorescent Protein and Firefly Luciferase Expressing Mouse Primary Macrophages for In Vivo Bioluminescence Imaging. PLoS ONE, 10(11), e0142736.
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6

Quantitative Analysis of Inflammatory Cytokines

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Cellular RNAs were extracted by using RNeasy RNA purification kit (Qiagen, Valencia, CA). RNAs were reverse transcribed into complementary DNA (cDNA) using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). Probes for 18s rRNA, TNF-α, IL-1β, IL-6, and MCP1 were purchased from Applied Biosystems. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed in an ABI 7900HT Sequencing Detection System (Applied Biosystems), using the 18s rRNA as the internal control. The -ΔΔCt relative quantitation method was used to evaluate gene expression level.
Lin T.H., Yao Z., Sato T., Keeney M., Li C., Pajarinen J., Yang F., Egashira K, & Goodman S.B. (2014). Suppression of wear particle induced pro-inflammatory cytokine and chemokine production in macrophages via NF-κB decoy oligodeoxynucleotide: A preliminary report. Acta biomaterialia, 10(8), 3747-3755.
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7

Gene Expression Analysis of Osteogenic Differentiation

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After 3 and 6 days of cocultures, RNA was extracted using Qiagen’s RNeasy Mini Kit (Valencia, CA) and reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). qRT-PCR was performed using TaqMan Gene Expression Master Mix and 18s, Tnf-α, Cd206, runt related transcription factor 2 (Runx2), alkaline phosphatase, liver/bone/kidney (Alpl), SMAD specific E3 ubiquitin protein ligase 2 (Smurf2), Transcriptional Co-Activator With PDZ-Binding Motif (Taz), Insulin growth factor-1 (Igf-1) and Vascular endothelial growth factor (Vegfa) probes on an ABI 7900HT Sequencing Detection System (all from Applied Biosystems). The GenBank accession numbers of the probe sequences are 18s: NR_003278, Tnf-α: NM_013693 and NM_001278601, Cd206: NM_008625 XM_001003164 XM_001003168, Runx2: NM_001146038, Alpl: NM_007431, Smurf2: NM_025481 XM_126673, Taz (Wwtr1): NM_001168281, Igf-1: NM_001111274, Vegfa: NM_001025250. 18s rRNA was the internal control. Relative gene expression was quantified with the comparative Ct method(14 ).
Córdova L.A., Loi F., Lin T.H., Gibon E., Pajarinen J., Nabeshima A., Lu L., Yao Z, & Goodman S.B. (2017). CCL2, CCL5 and IGF-1 Participate in The Immunomodulation of Osteogenesis during M1/M2 Transition In Vitro. Journal of biomedical materials research. Part A, 105(11), 3069-3076.
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8

Macrophage RNA Expression Profiling

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Cellular RNA from macrophages in the coculture system was extracted using the RNeasy RNA purification kit (Qiagen, Valencia, CA, USA). RNA was reverse transcribed into complementary DNA (cDNA) using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA). Probes for 18 s rRNA, TNF-α, IL-1Ra, Arg1, and CD206 were purchased from Applied Biosystems. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed in an ABI 7900HT Sequencing Detection System (Applied Biosystems), using 18 s rRNA as the internal control. The –ΔΔCt relative quantization method was used to evaluate the gene expression level.
Lin T., Pajarinen J., Nabeshima A., Lu L., Nathan K., Jämsen E., Yao Z, & Goodman S.B. (2017). Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis. Stem Cell Research & Therapy, 8, 277.
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9

Gene Expression Analysis of Osteogenic Markers

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RNA was extracted using Qiagen’s RNeasy Mini Kit (Valencia, CA, USA) and reverse transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed using TaqMan Gene Expression Master Mix and 18s, iNOS, TNFα, CD206, Arg1, osteocalcin (OC), osteopontin (OPN), and alkaline phosphatase (ALP) probes on an ABI 7900HT Sequencing Detection System (all from Applied Biosystems). The GenBank accession numbers of the probe sequences are 18s [NR_003278], iNOS [NM_010927;XM_001001508], TNFα [NM_013693; NM_001278601], CD206 [NM_008625;XM_001003164;XM_001003168], Arg1 [NM_007482], OC [NM_031368], OPN [NM_001204201;NM_001204202;NM_001204203;NM_001204233;NM_009263;XR_106288;XR_106289;XR_107716;XR_107717;XR_107718], and ALP [NM_007431]. 18s rRNA was the internal control. Relative gene expression was quantified with the comparative Ct method.
Loi F., Córdova L.A., Zhang R., Pajarinen J., Lin T.H., Goodman S.B, & Yao Z. (2016). The effects of immunomodulation by macrophage subsets on osteogenesis in vitro. Stem Cell Research & Therapy, 7, 15.
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10

Spinal Cord Gene Expression Analysis Post-Fracture

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At 4 weeks after fracture the rats were euthanized by CO2 inhalation and the ipsilateral spinal cord (L4,5 lumbar enlargement) was collected after behavioral testing and frozen immediately on dry ice. In mice the spinal cord was collected at 3 weeks post-fracture. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) and the purity and concentration were determined spectrophotometrically. Next, cDNA was synthesized from 1 μg RNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Real-time polymerase chain reactions (PCRs) were conducted using the SYBR Green PCR master mix (Applied Biosystems) and performed on an ABI 7900HT sequencing detection system (Applied Biosystems). To validate the primer sets used, we performed dissociation curves to document single product formation, and agarose gel analysis was conducted to confirm the size (Table 1). The data from real-time PCR experiments were analyzed as described in the manual for the ABI prism 7000 real-time systems. All results were confirmed by repeating the experiment 3 times.
Shi X., Guo T.Z., Wei T., Li W.W., Clark D.J, & Kingery W.S. (2015). Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to postfracture nociceptive sensitization. Pain, 156(10), 1852-1863.
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