Applied biosystems prism 7900 sequence detection system

Manufactured by Thermo Fisher Scientific

The Applied Biosystems Prism 7900 Sequence Detection System is a real-time PCR instrument designed for gene expression analysis and quantification. The system utilizes fluorescence detection technology to monitor the amplification of DNA samples in real-time during the PCR process.

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3 protocols using applied biosystems prism 7900 sequence detection system

Quantitative RT-PCR Analysis of CCR Gene Expression

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Total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Amplification of mRNA was performed and then it was transcribed from double-stranded cDNA using SuperScript™ III Reverse transciptase (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time RT-PCR was performed in triplicate in 96-well plates; each 10 μL reaction consisted of 5 μL of FAST Universal Master Mix (Applied Biosystems, Foster City, CA, USA), 0.5 μL of probe primer sets of CCR1 (Life Technologies, Carlsbad, CA USA), CCR2 (Life Technologies), CCR3 (Life Technologies), or ACTB (Applied Biosystems), 1.5 μL of ddH20, and 3 μL of template.
The real-time PCR analysis was performed on an Applied Biosystems Prism 7900 Sequence Detection System (Applied Biosystems).
Ct values were normalized for the deviations against ACTB serving as a housekeeping gene. The differential gene expression was estimated as: ΔCt = Ct_{(blank control)} − Ct_(mCCL7+), and fold-change = 2^(−ΔCt).

Kurzejamska E., Sacharczuk M., Landázuri N., Kovtonyuk O., Lazarczyk M., Ananthaseshan S., Gaciong Z, & Religa P. (2019). Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors. International Journal of Molecular Sciences, 20(3), 686.

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Genotyping of BDNF val66met Polymorphism

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DNA was extracted from white blood cells from blood samples collected from study participants with the Gentra Puregene blood kit (Qiagen, Germantown, MD). val66met (rs6265) genotyping was performed with a 5′-fluorogenic exonuclease assay (C__11592758_10) (TaqMan®, Applied Biosystems, Foster City, CA) or an ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) in duplicates by an individual blind to participants clinical status. Platinum® quantitative PCR SuperMix-UDG (Invitrogen, Carlsbad, CA) on a GeneAmp PCR system 9700 was employed for PCR amplification. Applied Biosystems Prism® 7900 sequence detection system and SDS 2.2 software (Applied Biosystems) were used for analysis of amplification products. In subsequent analyses individuals with val/met or met/met genotypes were combined (met carriers) and compared with individuals with the val/val genotype, because met/met carriers only occupy about 6% of the total sample.

Cao B., Bauer I.E., Sharma A.N., Mwangi B., Frazier T., Lavagnino L., Zunta-Soares G.B., Walss-Bass C., Glahn D.C., Kapczinski F., Nielsen D.A, & Soares J.C. (2016). Reduced hippocampus volume and memory performance in bipolar disorder patients carrying the BDNF val66met met allele. Journal of affective disorders, 198, 198-205.

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Quantitative Real-Time PCR Analysis

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Total mRNA was extracted from NCI-H292 and NHBE cells using TRI reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized from 2 lg total RNA using SuperScript II RNase Reverse Transcriptase (Invitrogen) in a 20-ll reaction containing Oligo-dT (12-18mer) primer, deoxynucleotide triphosphates (0Á5 mM), MgCl 2 (2Á5 mM), and dithiothreitol (5 mM). Reverse transcription was performed at 42°for 1 hr and followed by heat inactivation at 70°for 15 min. Synthesized cDNA was amplified for 30 cycles with thermostable DNA polymerase using Hot-start PCR premix (Bioneer, Daejeon, Korea). Quantitative real-time PCR was performed in a 20-ll reaction with 0Á5 ll cDNA, 0Á8 ll of each primer (10 pM), and 10 ll SYBR Green Master Mix (Applied Biosystems, Foster City, CA) using the Applied Biosystems Prism 7900 Sequence Detection System (Applied Biosystems). The PCR conditions were as follows: 2 min at 50°and 10 min at 95°, followed by 40 cycles of 95°for 30 seconds, 60°for 30 seconds and 72°for 30 seconds. The PCR primers were previously described. 30 (link) The specificity of amplification was confirmed by melting curve analysis and gel electrophoresis. Relative expression was evaluated using the comparative cycle threshold (2 ÀDDCt ) method and expressed as the mean AE SEM.

Kang J.H., Hwang S.M, & Chung I.Y. (2015). S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal-regulated kinase and nuclear factor-κB pathways. Immunology, 144(1).

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