Rt2 profiler pcr array

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The RT2 Profiler PCR Array is a real-time PCR-based system designed to analyze the expression of a focused panel of genes related to a specific biological pathway or disease state. It provides a comprehensive and efficient way to study gene expression profiles across multiple samples simultaneously.

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RT2 Custom Profiler PCR Arrays (4 citations) RT2 Profiler PCR Arrays kit (3 citations) RT2 Profiler PCR Arrays for Mouse Fibrosis (3 citations) RT2 Profiler PCR arrays for Human Antiviral Response (2 citations) Atherosclerosis RT2 Profiler PCR Arrays (2 citations) RT2 Profiler human adipogenesis PCR arrays (2 citations) Mouse Autophagy RT2 Profiler PCR Arrays (2 citations) RT2 Profiler ‘human angiogenesis’ PCR arrays (2 citations) Mouse DDR RT2 Profiler PCR Arrays (2 citations) Custom RT2 Profiler PCR Arrays and Primer Assays (2 citations)

622 protocols using rt2 profiler pcr array

Evaluating Gene Expression Changes in Bone and Adipose Tissue

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Tibiae (n=8/group) were pulverized with a mortar and pestle in liquid nitrogen and homogenized in Trizol (Life Technologies, Grand Island, NY). Total RNA was isolated according to the manufacturer’s protocol, and mRNA was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies). Gene expression related to osteoblast differentiation and function (Mouse “Osteogenesis” RT² Profiler PCR Array, PAMM-026ZE-4 and Mouse “Osteoporosis” RT² Profiler PCR Array, PAMM-170ZE-4) and adipocyte differentiation and function (Mouse “Adipogenesis” Signaling RT² Profiler PCR Array, PAMM-049ZE-4) was determined according to the manufacturer’s protocol (Qiagen, Valencia, CA). Gene expression was normalized to GAPDH. Relative quantification was determined (ΔΔCt method) using RT² Profiler PCR Array Data Analysis software version 3.5 (Qiagen). Fold-change was calculated using mice housed at 22°C as the control.

Iwaniec U.T., Philbrick K.A., Wong C.P., Gordon J.L., Kahler-Quesada A.M., Olson D.A., Branscum A.J., Sargent J.L., DeMambro V.E., Rosen C.J, & Turner R.T. (2016). Room Temperature Housing Results in Premature Cancellous Bone Loss in Growing Female Mice: Implications for the Mouse as a Preclinical Model for Age-Related Bone Loss. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA, 27(10), 3091-3101.

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Analyzing Senescence Genes in Mice

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The RT² profiler PCR array (QIAGEN, Hilden, Germany) was used to assess mouse senescence genes expression of 84 selected genes. RT² Profiler PCR Array results were analyzed with RT² Profiler PCR Array data analysis software, on the QIAGEN Web site [18 ], which gives us the average 2^–∆∆CT for mean ± SD for all wt and Prdx6^-/- groups. The system, also, automatically calculated the p value that we reported on the bar graphs.

Pacifici F., Della-Morte D., Piermarini F., Arriga R., Scioli M.G., Capuani B., Pastore D., Coppola A., Rea S., Donadel G., Andreadi A., Abete P., Sconocchia G., Bellia A., Orlandi A, & Lauro D. (2020). Prdx6 Plays a Main Role in the Crosstalk between Aging and Metabolic Sarcopenia. Antioxidants, 9(4), 329.

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Inflammatory and Immune Response Genes

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RT2 Profiler PCR Arrays were specific to genes involved in acute phase inflammatory and humoral immune responses, targeting cytokines, chemokines and their receptors, and regulators of cytokine metabolism and synthesis (>350 genes, see Supplementary Table S1). cDNA for each group (5 mice each) was pooled from equal amounts of RNA from each sample, totaling 1μg RNA for one plate. The SABiosciences RT² Profiler PCR Array (PAMM-3803E-12) was conducted according to the manufacturer’s protocol with the ABI 7900HT (Applied Biosystems). Data was analyzed using the SABiosciences RT² Profiler PCR Array data analysis program. Genes with differences of at least a 2-fold change were included in lists of differentially expressed genes.

Shea A.A., Heffron C.L., Grieco J.P., Roberts P.C, & Schmelz E.M. (2024). Obesity modulates the cellular and molecular microenvironment in the peritoneal cavity: implication for ovarian cancer risk. Frontiers in Immunology, 14, 1323399.

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Oxidative Stress and Antioxidant Defense in Mouse Cochlear Tissues

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The Mouse Oxidative Stress and Antioxidant Defense RT² Profiler™ PCR array (SABiosciences Corp., Valencia, CA) was used to measure the alteration of gene expression in the cochlear tissues from WT- and Cmah-null mice. The RT² Profiler™ PCR Array (SABiosciences Corp.) is a commercially available set of optimized RT-qPCR primer assays on a 96-well plate. Total RNA was then treated with DNase I, reverse-transcribed using an RT² First Strand Kit (Qiagen), and brought to a final volume of 120 μL. cDNA from individual samples was used as a template for the PCR array, according to the array instructions, using SYBR green on an ABI ViiA^TM 7 system (Applied Biosystems). Data were analyzed using SABiosciences RT2 Profiler PCR Data Analysis software, available at http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php, and were considered significant at >4-fold change. Relative quantitation for each gene was determined by normalization to 5 housekeeping genes (Actb, B2m, Gapdh, Gusb, and Hsp90ab1), comparing the WT and Cmah-null groups by using the 2-ΔΔCt method.

Kwon D.N., Park W.J., Choi Y.J., Gurunathan S, & Kim J.H. (2015). Oxidative stress and ROS metabolism via down-regulation of sirtuin 3 expression in Cmah-null mice affect hearing loss. Aging (Albany NY), 7(8), 579-592.

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Oxidative Stress and Antioxidant Gene Expression

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The Human Oxidative Stress and Antioxidant Defense RT² Profiler™ PCR array (SABiosciences Corp., Valencia, CA) was used to measure the alteration of gene expression in the platelet RNA isolated from the different age cohorts. The RT² Profiler™ PCR Array (SABiosciences Corp.) is a commercially available set of optimized qPCR primer assays on a 96-well plate. Total RNA was treated with DNase I, reverse-transcribed using an RT² First Strand Kit (Qiagen), and brought to a final volume of 120 μl. cDNA was pre-amplified using RT2 Nano PreAmp cDNA Synthesis Primer Mix Human Oxidative Stress kit according to manufacturer's instructions (SabBioscience, Qiagen). Amplified cDNA from individual samples was used as a template for the PCR array, according to the array instructions, using SYBR green on CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories). Data was analyzed using SABiosciences RT2 Profiler PCR Data Analysis software, available at http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php, and were considered significant at >2-fold change. Relative quantitation for each gene was determined by normalization to the housekeeping genes included with the array by using the 2-ΔΔCt method and represented as fold change over the Young Control group. The Array was performed in n = 3 samples for each age cohort.

Jain K., Tyagi T., Patell K., Xie Y., Kadado A.J., Lee S.H., Yarovinsky T., Du J., Hwang J., Martin K.A., Testani J., Ionescu C.N, & Hwa J. (2019). Age associated non-linear regulation of redox homeostasis in the anucleate platelet: Implications for CVD risk patients. EBioMedicine, 44, 28-40.

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Profiling Gene Expression Changes in OPCs

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Total RNA was extracted by means of RNeasy Micro kit (Qiagen) following the manufacturer's protocol. The PCR arrays "Wnt signaling targets" (PARN-243ZD), "Extracellular Matrix & Adhesion Molecules" (PARN-013ZD) and "Gap Junction" (PARN-144Z) were used to identify genes differentially expressed in OPCs after miR-125a-3p over-expression compared to negative control (See Supplementary material for the full gene list).
For each PCR array, cDNA synthesis was performed starting from 500 ng of DNase pre-treated total RNA using RT2 First Strand Kit (Qiagen), following manufacturer's protocol. RT2 Profiler PCR Array (SABiosciences) and RT2 SYBRgreen Mastermix (Qiagen) were used to measure gene expression levels. Each array includes five housekeeping genes, that enable normalization of data, a genomic DNA control, that specifically detects genomic DNA contamination, a reverse transcription control, that tests the efficacy of the reverse-transcription reaction and a positive PCR control, that tests the efficacy of the polymerase chain reaction itself. Data were analysed by RT2 Profiler PCR Array data analysis center v. 3.5 (Qiagen).

Marangon D., Abbracchio M.P, & Lecca D. (2021). Pathway-Focused Profiling of Oligodendrocytes Over-Expressing miR-125a-3p Reveals Alteration of Wnt and Cell-to-Cell Signaling. Cellular and molecular neurobiology, 41(1).

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Evaluating Cell Cycle and Apoptosis Genes

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Cell cycle and apoptosis pathway genes were evaluated using specific 96-well plate RT² Profiler PCR Arrays from Qiagen (Qiagen, Germantown, MD, USA). Mouse cell cycle (PAMM-020ZA, Qiagen) and mouse apoptosis (PAMM-012Z, Qiagen) panels were used to design these arrays. The RT² Profiler PCR Array included controls for genomic DNA contamination, reverse transcription efficiency, real-time PCR efficiency and housekeeping genes β-glucuronidase (Gusb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), heat shock protein 90 alpha (cytosolic), class B member (Hsp90ab1) and β-2 microglobulin (B2m). One µg of total RNA was converted to cDNA using Qiagen RT² first strand kit (Qiagen, Catalog# 330404). RT-qPCR analysis of Qiagen RT² Profiler PCR Array was performed using SYBR green with Rox according to the manufacturer’s instructions. All raw data were uploaded to Qiagen data analysis website (https://geneglobe.qiagen.com). Samples from five separate experiments were analyzed.

Srivastava T., Joshi T., Heruth D.P., Rezaiekhaligh M.H., Garola R.E., Zhou J., Boinpelly V.C., Ali M.F., Alon U.S., Sharma M., Vanden Heuvel G.B., Mahajan P., Priya L., Jiang Y., McCarthy E.T., Savin V.J., Sharma R, & Sharma M. (2021). A mouse model of prenatal exposure to Interleukin-6 to study the developmental origin of health and disease. Scientific Reports, 11, 13260.

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Tissue-specific Gene Expression Analysis

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Total RNA was extracted from liver, epWAT, BAT, gastrocnemius and terminal ileum (4 mice/group) using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen) following the manufacturer’s instructions. A total of 10 ng of cDNA was pipetted into each well of a 96-well Custom RT² Profiler PCR Array [CAPM13349, RT² Profiler PCR Array, Qiagen (http: www.qiagen.com/it/products/catalog/assay-technologies/real-time-pcr-and-Real-time PCR-reagents/rt2-qpcr-primer-assays/l)] and amplified following the manufacturer’s instructions in StepOnePlus instrument (Applied Biosystems). This RT² PCR array was settled up to assess a total of 75 genes: 30 genes for liver, 6 for gastrocnemius, 6 for intestine, 22 for epWAT and 11 for BAT; 2 housekeeping genes were used for each tissue for data normalization. The RT² Profiler PCR Array also includes control elements for: Genomic DNA contamination detection, RNA sample quality and General PCR performance. Data analysis is based on the ΔΔC_T method with normalization of the raw data to either housekeeping genes.

Carino A., Cipriani S., Marchianò S., Biagioli M., Scarpelli P., Zampella A., Monti M.C, & Fiorucci S. (2017). Gpbar1 agonism promotes a Pgc-1α-dependent browning of white adipose tissue and energy expenditure and reverses diet-induced steatohepatitis in mice. Scientific Reports, 7, 13689.

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Quantitative PCR analysis of gene expression

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For standard qPCR, total RNA was extracted from frozen tissue, reverse transcribed and performed as previous described.^{9 (link)} Gene expression was normalised to housekeeping gene (β-actin or TATA-binding protein) and then expressed as fold change of SLD-fed WT mice using the 2^-ΔΔCT method. Primer sequences are provided in Supplementary Table S1.
For RT² Profiler PCR array (Qiagen/SA Biosciences, Valencia, CA, USA) analysis, first-strand cDNA was synthesised from total RNA using the RT² First strand cDNA synthesis kit (Qiagen). The RT² Profiler PCR array system was set up, run and analysed on the Viaa 7 Real-Time PCR System (Thermo Fisher Scientific Inc., Wilmington, DE, USA) according to the manufacturer's instructions. RT² Profiler PCR arrays used were: mouse fatty liver (#PAMM-157Z); mouse glucose metabolism (#PAMM-006Z); mouse insulin resistance (#PAMM-156Z, Qiagen/SA Biosciences). Gene lists and functional grouping are provided in Supplementary Data S1-S3. PCR arrays were 96 × 4 format allowing for four-independent samples to be run on each array. Data were processed using RT² Profiler PCR Array Data Analysis version 3.5 online software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).

Wilson C.H., Nikolic A., Kentish S.J., Keller M., Hatzinikolas G., Dorstyn L., Page A.J, & Kumar S. (2017). Caspase-2 deficiency enhances whole-body carbohydrate utilisation and prevents high-fat diet-induced obesity. Cell Death & Disease, 8(10), e3136-.

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Quantifying Bovine Inflammatory Cytokines

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The Cow Inflammatory Cytokines & Receptors RT² Profiler™ PCR Array (Qiagen) was used for the quantitative real-time RT-PCR (RT-qPCR) analysis of the ICLN of all sheep. Total RNA was extracted as described above and each sample of RNA was reverse transcribed using a RT² First Strand Kit according to the manufacturer’s protocol (Qiagen). Real-time PCR was performed on each cDNA sample using the RT² Profiler™ PCR Array with the RT² SYBR Green/ROX FAST Mastermix in a Rotor-Gene Q cycler (Qiagen). The cycling profile was performed at 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Melting curve analysis of qPCR products confirmed the absence of secondary product. RT² Profiler PCR Array Data Analysis version 3.5 software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) was used for data analysis. Spearman rank-order correlation analysis was carried out using GraphPad Prism 6.07 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).

Gossner A., Watkins C., Chianini F, & Hopkins J. (2017). Pathways and Genes Associated with Immune Dysfunction in Sheep Paratuberculosis. Scientific Reports, 7, 46695.

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