Insulin, progesterone, putrescine, selenium, transferrin, anti-NGF, NGF, poly-d -lysine, NSC663284 and NSC95397 were purchased from Sigma (St Louis, MO, USA). Cell culture media DMEM, DMEM-F12, RPMI-1640, Neurobasal, B27, antibiotics, Lipofectamine 2000, Alexa Fluor, cDNA synthesis kit, real-time PCR kit and serum were purchased from Invitrogen (Life technologies, Grand Island, NY, USA). Cell culture dishes, plates and flasks were purchased from BD Falcon, Corning (Corning, NY, USA). Aβ(1–42) and Aβ(42-1) were purchased from American Peptide (Sunnyvale, CA, USA). ECL reagent and PVDF membrane were purchased from GE Healthcare (Buckinghamshire, UK). The PCR kits were purchased from Takara (Shiga, Japan), Fermentas (Waltham, MA, USA). Anti-Cdc25A, anti-Actin and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phospho-Rb antibody was purchased from Cell Signaling Technologies (Denver, MA, USA). AKT inhibitor II, JNK inhibitor II and U0126 were purchased from Calbiochem (Darmstadt, Germany). Primers were purchased from IDT DNA (Gurgaon, Haryana, India). Brain tissues of AβPPswe-PS1de9 mice and control littermates were a kind gift from Dr. Anant B. Patel (Council of Scientific and Industrial Research-Centre for Cellular and Molecular Biology (CSIR- CCMB)), Hyderabad, India.
Real time pcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Real-time PCR kit is a laboratory instrument used for the quantitative detection and analysis of nucleic acid sequences. It utilizes the polymerase chain reaction (PCR) technique to amplify and detect specific DNA or RNA targets in a sample. The kit provides the necessary reagents and consumables required to perform real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rna extraction kit, by Tiangen Biotech (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Selenium, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
Anti cdc25a, by Santa Cruz Biotechnology (1 mentions)
Nsc95397, by Merck Group (1 mentions)
Dmem cell culture media, by Thermo Fisher Scientific (1 mentions)
Pcr kit, by Takara Bio (1 mentions)
Anti phospho rb antibody, by Cell Signaling Technology (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Nerve growth factor (ngf), by Merck Group (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Anti ngf, by Merck Group (1 mentions)
19 protocols using real time pcr kit
1
In Vitro Neurodegeneration Assay
2
M1 Macrophage Gene Expression Analysis
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THP-1 cells were treated with different doses of LLLT and total RNA was isolated from cells immediately (t = 0) or 6 hours after LPS stimulation. Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. Three μg of RNA from each sample was then reverse-transcribed into first-strand cDNA in 20 μL of reaction mixture using the SuperScript First-Strand Synthesis System with the Real-time PCR kit (Invitrogen). Measurements were performed by an ABI PRISM 9700 HT sequence detection system (Applied Biosystems, Foster City, CA) using a predeveloped Taqman probe/primer combination for M1-related genes and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from the same cDNA samples. Taqman PCR was performed in 10 μL using AmpliTaq Gold polymerase and the universal master mix (Applied Biosystems). Threshold cycle numbers were transformed using the comparative threshold cycle and relative value methods according to the manufacturer's recommendation and expressed relative to G3PDH, which is used as a housekeeping gene by multiplexing single reactions. The M1-related cytokine and chemokine genes are as follows: CCL2/MCP-1, CXCL10/IP-10, and TNF-α.
3
Quantitative Gene Expression Analysis
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The total RNA was isolated using RNA extraction kit (TIANGEN) and reverse transcribed into cDNA using reverse transcription kit (ABI). Quantitative real‐time PCR analysis was performed using real‐time PCR kit (ABI). The relative mRNA expression levels of IDO, IL‐2, IFN‐γ, IL‐10, IL‐4 and TNF‐αwere normalized with the β‐actin in the same sample. The thermal cycler parameters for the amplification of these genes were as follows: 1 cycle at 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds, 60°C for 15 seconds and 72°C for 30 seconds. Gene expression was evaluated by the 2−ΔΔCt method. The sequences of RT‐PCR primers are the following (5′‐3′, Table 1 ).
4
Quantitative Real-Time PCR Analysis
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The total RNA was isolated using RNA extraction kit (TIANGEN) and reverse transcribed into cDNA using reverse transcription kit (ABI). Quantitative real‐time PCR analysis was performed using real‐time PCR kit (ABI). The relative mRNA expression levels of STAT3, MCL1, PMAIP1, SOD2, FOXO3, FOS, and FKBP5were normalized with the GADPH in the same sample. The thermal cycler parameters for the amplification of these genes were as follows: one cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 s, 60°C for 15 s and 72°C for 30 s. Gene expression was evaluated by the 2−ΔΔCt method. The sequences of RT‐PCR primers are the following (5–3′, Table 1 ).
5
RNA Extraction and RT-qPCR Analysis
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Total RNAs were extracted from cells or lung tissue using the Trizol (Ambion,127703) reagent according to the manufacturer’s instructions. cDNA was synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher, K1622). The Real-Time PCR Kit (Applied biosystems, A25742) reactions were run in duplicate for each sample, and transcript levels of target genes were normalized to that of β-actin. The primer sequences used are shown in Table 1 .
6
Quantitative mRNA Analysis of Adipose Tissue
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Total RNA was extracted from epididymal adipose tissue and SVFs of epididymal fat tissue, liver, and macrophages using TRI reagent (Molecular Research Center, Cincinnati, OH) and reverse transcribed, as described previously (24 (link)). For detecting various mRNA transcripts, a commercially available real-time PCR kit was used (Applied Biosystems, Foster City, CA), and all data were normalized to constitutive rRNA (18S) values. The Applied Biosystems 7700 sequence detector was used for amplification of target sequences, and quantitation of differences between treatment groups was performed using the comparative threshold cycle method.
7
Quantifying Gene Expression via RT-PCR
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Total RNA was isolated from NRK-52E cells and frozen renal tissues using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Equal amounts of RNA (2 μg) were reverse-transcripted into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche; IN, USA). The specific primers for GSTA3, α-SMA, E-cadherin, Fibronectin, Collagen I and β-actin were designed from their GenBank sequences, synthesized by Bio Basic (Shanghai, China), and are listed in Table 1 . The RT-PCR quantitation for individual target mRNA expression was performed on an ABI Model 7500 Sequence Detector (Applied Biosystems, Foster City, CA, USA) using a TaKaRa real-time PCR kit. The amplified PCR products were quantified by measuring the target and β-actin mRNA calculated cycle thresholds (CT). The amount of specific mRNA in each sample was calculated from the standard curve and normalized with β-actin [19 (link)].
8
Polypeptide-SNA Regulation of MYC Expression
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Example 14
HEK293T cells were treated with an exemplary polypeptide alpha (FQGRLLRLYILP; SEQ ID NO: 19) linked to an exemplary SNA (e.g. 5′-AAGTAAGTGTGCCCTCTACTGGCAGCAGAGATCAT-3′; SEQ ID NO: 20 resulting in a polypeptide-SNA of, e.g. SEQ ID NO: 10) engineered to bind to sequences flanking a MYC gene sequence (with or without fluorescent dye). Additional exemplary polypeptides are listed in Table 1. At 72 hours post-treatment, cells were harvested for RNA extraction and cDNA was synthesized (Thermo Fisher Scientific) according to manufacturer's protocols. cDNA was used as a template for quantitative real-time PCR.
MYC-specific quantitative PCR probes/primers were multiplexed with internal control quantitative PCR probes/primers and gene expression was subsequently analyzed by a real time PCR kit (Applied Biosystems, Thermo Fisher Scientific).
Cells treated with SNAs that are complementary to sequences proximal to the MYC gene showed reduction in MYC expression indicating translocation of a provided polypeptide and linked SNA.
Preliminary studies with an exemplary polypeptide alpha conjugated to SNAs indicate no adverse effects of peptides on ability of SNAs to reduce MYC expression levels.
9
MYC Expression Regulation by SNA-Conjugated Peptides
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Example 15
HEK293T cells are treated with exemplary polypeptide betas and/or gammas linked to SNAs (exemplary peptides are listed in Table 1) engineered to bind to sequences flanking a MYC gene (with or without fluorescent dye). At 72 hours post-treatment, cells are harvested for RNA extraction and cDNA is synthesized (Thermo Fisher Scientific) according to manufacturer's protocols. cDNA is used as a template for quantitative real-time PCR.
MYC-specific quantitative PCR probes/primers are multiplexed with internal control quantitative PCR probes/primers and gene expression is subsequently analyzed by a real time PCR kit (Applied Biosystems, Thermo Fisher Scientific).
Cells treated with SNAs proximal to the MYC gene are expected to show reduction in MYC expression indicating translocation of the peptide and linked SNA.
Results of studies using exemplary polypeptide betas or gammas conjugated to SNAs will indicate no adverse effects of provided polypeptides on ability of SNAs to reduce MYC expression levels.
10
Real-time PCR Amplification and Analysis
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Real-time PCR was performed using real-time PCR kit (Applied Biosystems, USA). The reaction parameters were: 35 cycles of 94 °C for 5 min, 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s and 72 °C for 10 min. Real-time PCR primer sequences were also listed as in Table 1 .
After each template and gene amplification, a program was set: 95 °C for 15 s, 62 °C for 20 s, temperature 62 °C slowly increased to 95 °C for 15 s within 20 min. Continuously the fluorescent signal of sample was collected in the process of climbing to get the melting curve, and the melting curve was available through quantitative real-time PCR own analysis software.
After each template and gene amplification, a program was set: 95 °C for 15 s, 62 °C for 20 s, temperature 62 °C slowly increased to 95 °C for 15 s within 20 min. Continuously the fluorescent signal of sample was collected in the process of climbing to get the melting curve, and the melting curve was available through quantitative real-time PCR own analysis software.
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