PCR-based microarrays for evaluating the expression of genes mediating the inflammatory response were performed using the human inflammatory cytokines and receptors RT2 Profiler TM PCR array (Qiagen, PAHS-011ZE-4); PCR-based microarrays for evaluating the expression of genes in breast cancer cell lines were performed using the Breast cancer PCR array RT2 Profiler TM PCR array (Qiagen, PAHS-131Z-4) and the Tumor Metastasis PCR array RT2 Profiler TM PCR array (Qiagen, PAHS-028Z). The arrays were configured in a 384-well plate consisted of a panel of 92 genes and 4 endogenous genes. Reverse transcription was performed using the RTC First Strand Kit (Qiagen, 330401) and qPCR was performed using RTC SYBR Green/ROX PCR Master mix (Qiagen, 330521), and the raw data were analyzed by the GeneGlobe Data Analysis Center (www.qiagen.com ) according to the manufacturer’s instructions.
Rt2 profilertm pcr array
Manufactured by Qiagen
Sourced in Germany, United States
The RT2 ProfilerTM PCR array is a real-time PCR-based gene expression profiling system. It enables the simultaneous analysis of the expression levels of a focused panel of genes related to a specific biological pathway or disease state. The core function of the RT2 ProfilerTM PCR array is to provide a comprehensive and accurate assessment of gene expression patterns in a given sample.
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Lab products found in correlation
Rt2 first strand kit, by Qiagen (6 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Geneglobe data analysis center, by Qiagen (3 mentions)
Abi prism 7000, by Thermo Fisher Scientific (2 mentions)
Sa biosciences rt2 qpcr master mix, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rt2 sybr green rox qpcr mastermix, by Qiagen (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Cfx96, by Bio-Rad (2 mentions)
Steponeplus real time pcr, by Thermo Fisher Scientific (1 mentions)
Rnase free water, by Qiagen (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Imagemastertm 2d platinum 7, by GE Healthcare (1 mentions)
Rna extraction kit, by Qiagen (1 mentions)
Gapdh primer, by Qiagen (1 mentions)
Cdna kit, by Thermo Fisher Scientific (1 mentions)
Caspase 3 7 activity assay kit, by Promega (1 mentions)
17 protocols using rt2 profilertm pcr array
1
Comprehensive Gene Expression Profiling of Inflammatory and Breast Cancer Pathways
2
Cisplatin-Induced DNA Damage Signaling Pathway
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The relative mRNA expressions after cisplatin stimulation in IMC-3CR were analyzed using an RT2 ProfilerTM PCR array (Human DNA Damage Signaling Pathway, Qiagen) according to the manufacturer’s protocol34 (link). Briefly, IMC-3CR cells (5 × 105) were placed in RPMI 1640 supplemented with 10% heat-inactivated FCS. Cells with or without cisplatin (1 μg/ml) were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 6 h. After incubation, cells were washed with PBS, total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized through RT performed with an RT2 First Strand Kit (Qiagen) according to the manufacturer’s protocol. The cDNA was applied to the PCR array and real-time PCR performed on a Sequence Detection System (ABI PRISM® 7000; Life Technologies Corporation) using PCR master mix (SA Biosciences RT2 qPCR Master Mix; Qiagen) for SYBR Green detection for each reaction. Samples were amplified with a precycling hold at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing for 1 min at 60 °C. The PCR array results were uploaded to the RT2 Profiler PCR Array Data Analysis website (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php ). The alteration of mRNA expression was analyzed using ΔΔCt method.
3
Epithelial-Mesenchymal Transition PCR Profiling
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The human Epithelial to Mesenchymal Transition RT2 ProfilerTM PCR Array (PAHS-090ZC-2) was purchased from Qiagen (Venlo, Netherlands). The recommended RT2 First Strand Kit (330401) and RT2 SYBR® Green ROCTM qPCR Master Mix (330520) were used as per the manufacturer’s instructions.
4
Quantitative Gene Expression Analysis
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Total RNA was isolated from tissue samples stabilized in RNA later using Qiagen RNeasy Mini Kit as per manufacturer’s guidelines. For RT2 ProfilerTM PCR array 0.5ug RNA was converted to cDNA using RT2 First Strand Kit (Qiagen) as per manufacturer’s guidelines. cDNA was then used in RT2 ProfilerTM PCR array Human Chemokines and Receptors (cat. No. PAHS-022Z). The array comprised of 84 target genes, 5 houskeeping genes, 3 Positive PCR controls and 3 Reverse Transcriptase Controls. Array was setup as per manufacturer’s guidelines on StepOne Plus RealTime PCR (Applied Biosystems). Results were analysed using Qiagen’s online webportal –Data Analysis Center.
5
Cytokine and Chemokine Profiling by PCR Array
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Expression levels of cytokine and chemokine mRNAs were analyzed using PCR array assays. Total RNA was isolated from cells using the High Pure RNA isolation Kit (Roche, Berlin, Germany) with a DNase I treatment during the purification and elution in 50 μl of RNase-free water (Qiagen). Reverse transcription was performed with the RT2 First strand Kit (Qiagen, Courtaboeuf, France), and the cDNA samples were analyzed in duplicate using a RT2 Profiler TM PCR array (Qiagen). SYBR green PCR conditions were 95°C for 10 min and 40 cycles of 95°C for 15 s, and 60°C for 1 min using 84 human inflammatory and receptor genes. The potential mRNAs were chosen and then confirmed by RT-qPCR.
The genes that contributed in each axis in the PCA were as follows:F1 = CCL1, 2, 4, 5, 7 13, 15, 17, 20, 22, CSF1, CX3CL1, CXCL 1 to 3, 5, 8 to 11, FASLG, IFNG, IL10RA, IL10RB, IL15, IL1a, IL1b, IL7, NAMPT, TNFSF4, 10, 11, 13, 13B, and VEGFA. F2 = AIMP1, C5, CCL1, 2, 13, 17, 23, CRR1, 2, 3, 4, 5, CSF1, CX3CR1, CXCR2, IL10RA, I10RB, IL15, LTA, LTB, MIF, SPP1, TNF, TNFSF4, 10, 11, 13, and 13B. F3 = CCL17, 23, CCR5, CX3CR1, IL10RA, IL5, IL9, MIF, and OSM.
The genes that contributed in each axis in the PCA were as follows:
6
Mesenchymal Stem Cell RNA Profiling
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Total RNAs from hMSCs were extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The 84 genes representing characteristics of hMSCs were quantitatively analysed using a commercial qPCR array kit (RT2 ProfilerTM PCR array: Human Mesenchymal Stem Cells, Qiagen, Hilden, Germany) following the manufacturer’s instructions.
7
Profiling LSC Transcriptional Pathways
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The cells were analyzed and sorted using the MoFlo FACS system (XDP, Beckman), and LSCs were identified inside the area of CD34+ and CD38-. FACS and trypan blue staining detected the enrichment of LSCs. Human Signal Transduction Pathway FinderTM RT2 ProfilerTM PCR Array (PAHS-014A, Qiagen, USA) was used to screen the differences in 84 key genes that are representative of 18 different signal transduction pathways between LSCK562 and LSCK562/ADM. Additional details are provided in Supplementary Detailed methods.
8
Comparative Analysis of Ruptured and Non-Ruptured Proteomics
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Baseline demographic data were expressed as mean±SD for continuous variables, and as frequency for categorical variables. Any significant differences between the experimental and control groups were assessed using t-tests, chi-square tests, and Fisher exact tests, as appropriate.
Differences between the ruptured and non-ruptured groups were evaluated using ImageMasterTM 2D Platinum 7.0 (GE Healthcare, Uppsala, Sweden) software for proteomics and free PCR array data analysis software provided by the manufacturer of the RT2 ProfilerTM PCR array (QIAGEN) for qPCR (according to the manufacturer’s instructions). The t-tests and Fisher exact tests were also used to analyze the IHC and ELISA data, respectively. The P-values <0.05 were considered statistically significant. All statistical analyses were conducted using the SPSS version 22.0 package for Windows (IBM Co., Armonk, NY, USA).
Differences between the ruptured and non-ruptured groups were evaluated using ImageMasterTM 2D Platinum 7.0 (GE Healthcare, Uppsala, Sweden) software for proteomics and free PCR array data analysis software provided by the manufacturer of the RT2 ProfilerTM PCR array (QIAGEN) for qPCR (according to the manufacturer’s instructions). The t-tests and Fisher exact tests were also used to analyze the IHC and ELISA data, respectively. The P-values <0.05 were considered statistically significant. All statistical analyses were conducted using the SPSS version 22.0 package for Windows (IBM Co., Armonk, NY, USA).
9
Neurogenesis Genes Expression in TBI
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84 genes related to neurogenesis were studied using RT2 ProfilerTM PCR Array (Qiagen). At day 3, RNA was extracted from penumbral cortex from TBI injured (N = 4) or uninjured (N = 4) rats, empirically amplified and undergone RT-PCR using commercial available primer-sets according to manufacturer’s protocol.
10
Analyzing Cisplatin-Induced DNA Damage Response
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The relative mRNA expressions of genes involved in DNA damage activities were analyzed following cisplatin stimulation of A375 using an RT2 ProfilerTM PCR array (Human DNA Damage Signaling Pathway, Qiagen) in adherence to manufacturer’s instructions36 (link). In brief, cells treated with or without cisplatin (4 μM) were incubated at 37 °C under a humidified atmosphere containing 5% CO2. After incubation, washing cells with PBS, total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized by RT-PCR using an RT2 First Strand Kit (Qiagen) according to the manufacturer’s protocol. The cDNA was applied to the Profiler PCR array and real-time PCR was performed on a Sequence Detection System (ABI PRISM® 7000; Life Technologies Corporation) using PCR master mix (SA Biosciences RT2 qPCR Master Mix; Qiagen) for SYBR Green detection for each reaction. Samples were amplified with a precycling hold at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing for 1 min at 60 °C. Changes in mRNA expression were analyzed using ∆∆Ct method. The expression levels were quantified relative to the values obtained for some housekeeping genes (ACTB, B2M, GAPDH, HPRT1, and RPL13A).
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