Taqman openarray real time pcr master mix

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TaqMan™ OpenArray™ Real-Time PCR Master Mix is a reagent formulation designed for use in real-time PCR applications. It is optimized for use with the OpenArray™ real-time PCR platform.

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16 protocols using taqman openarray real time pcr master mix

Customized Cardiovascular miRNA Profiling

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The screening was performed on a selected cardiovascular disease-associated miRNA panel using an OpenArray Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol for liquid samples. In particular, we used custom plates allowing the detection of 112 miRNAs (Table S2). Starting from 2 µL of total RNA, miRNA retrotranscription was conducted using TaqMan Advanced miRNA cDNA synthesis (Life Technologies, Waltham, MA, USA), according to the manufacturer’s protocol. A mixture composed of 2.5 µL of each amplified cDNA and 2.5 µL of TaqMan OpenArray Real-Time PCR master mix (Applied Biosystems, Waltham, MA, USA) was dispensed on an OpenArray plate using the AccuFill automated sample loading system (Applied Biosystems, Waltham, MA, USA) and run using a QuantStudio 12k Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Data analysis was conducted using cloud-based Thermo Fisher Scientific proprietary software (Thermo Fisher cloud, https://apps.thermofisher.com/apps/). Samples presenting a Ct value >35, an amplification score (Amp Score) <1.24 and a Ct confidence value <0.8 were excluded. The expression data for each sample are presented in Table S3. Normalization was conducted using the Global Mean Normalization method implemented by the software.

Gioffré S., Chiesa M., Cardinale D.M., Ricci V., Vavassori C., Cipolla C.M., Masson S., Sandri M.T., Salvatici M., Ciceri F., Latini R., Staszewsky L.I., Pompilio G., Colombo G.I, & D’Alessandra Y. (2020). Circulating MicroRNAs as Potential Predictors of Anthracycline-Induced Troponin Elevation in Breast Cancer Patients: Diverging Effects of Doxorubicin and Epirubicin. Journal of Clinical Medicine, 9(5), 1418.

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Quantitative VAMP2 Gene Expression in PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient (Lympholyte-H, Cedarlane, Canada). Total RNA was extracted from PBMCs using the Chomczynski and Sacchi’s modified method (Chomczynski and Sacchi, 2006 (link)). Two micrograms of total RNA were reverse-transcribed using the SuperScript VILOTM cDNA Synthesis Kit (Invitrogen by Thermo Fisher Scientific, Massachusetts, United States). Quantitative PCRs were performed in the OpenArray^® system QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems by Thermo Fisher Scientific, Massachusetts, United States). For the VAMP2 gene expression analysis, qPCR reactions based on TaqMan probes Hs00360269_m1 (Thermo Fisher Scientific) were performed using high-performance OpenArray^® chip. Three genes have been selected as endogenous according to their stable expression in human cells (GAPDH, ACTB and 18S) and included into the OpenArray^® chip. One hundred and twenty ng of every cDNA sample (1.2 μL of each) were mixed with 1.3 μL of PCR-grade water and 2.5 μL of TaqMan™ OpenArray^® Real-Time PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Massachusetts, United States). Samples were loaded in duplicate into Open-Array^® plates. For gene expression analysis, Ct values were obtained using the Thermo Fisher ConnectTM (Thermofisher) online application, and the Relative Quantification (RQ) software.

Costa A.S., Ferri E., Guerini F.R., Rossi P.D., Arosio B, & Clerici M. (2022). VAMP2 Expression and Genotype Are Possible Discriminators in Different Forms of Dementia. Frontiers in Aging Neuroscience, 14, 858162.

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Quantitative Analysis of ncRNA and miRNA

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Total RNA in OS-732 cells was extracted using RiboPure™ RNA Purification Kit (Invitrogen) according to the manufacturer’s instruction. The cDNA was reversely transcribed using GoScript™ Reverse Transcriptase System (Promega, Madison, WI, USA) in line with the supplier’s instruction. The TaqMan^® Noncoding RNA Assay (Applied Biosystems, Foster City, CA, USA) was used for real-time PCR analysis to detect the expression of CAT104. The TaqMan™ OpenArray™ Real-Time PCR Master Mix (Applied Biosystems) was performed to measure the expression of miR-381. For testing the expression of zinc finger E-box-binding homeobox 1 (ZEB1), Power SYBR Green Master Mix (Applied Biosystems) was used. All experiments were performed three times, and the relative expression level was calculated using the 2^−ΔΔCt method29 (link). The expression of GAPDH acted as the internal control for testing the expressions of CAT104 and ZEB1. The expression of U6 acted as the internal control for testing the expression of miR-381.

Xia B., Wang L., Feng L., Tian B., Tan Y, & Du B. (2018). Knockdown of Long Noncoding RNA CAT104 Inhibits the Proliferation, Migration, and Invasion of Human Osteosarcoma Cells by Regulating MicroRNA-381. Oncology Research, 27(1), 89-98.

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RNA Extraction and qRT-PCR Analysis

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RNA was purified using RNeasy Micro Kit (Qiagen, Austin, TX, USA) according to manufacturer’s protocol. cDNA was synthesized using iScript™ Reverse Transcription Supermix (Bio-rad, Herlev, Denmark). Quantitative real-time PCR was carried out on ViiA-7 using 1 ng of cDNA mixed with TaqMan™ OpenArray™ Real-Time PCR Master Mix (Applied Biosystems™, Thermo Fisher Scientific, Bleiswijk, The Netherland) and TaqMan primers (Table S5). Gene expression was analyzed by the 2 ΔCT method with beta actin (ACTB) as endogenous control.

Torz L., Niss K., Lundh S., Rekling J.C., Quintana C.D., Frazier S.E., Mercer A.J., Cornea A., Bertelsen C.V., Gerstenberg M.K., Hansen A.M., Guldbrandt M., Lykkesfeldt J., John L.M., Villaescusa J.C, & Petersen N. (2022). NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model. International Journal of Molecular Sciences, 23(6), 3260.

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Quantification of mRNA Expression

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For quantification of mRNA, RNA was extracted from 0.2–5.0 × 10⁶ splenocytes or ≈20mg frozen lung, kidney, brain, liver, heart, muscle, or small intestine using RNeasy Plus Universal Kits (Qiagen). cDNA was synthesized from the total RNA with the SuperScript™ VILO™ Master Mix (Thermo Fisher) with the following protocol: 25°C for 10 min, 42°C for 60 min, 85°C for 5 min. Transcript expression was measured with the appropriate primers using a QuantStudio 3 Real-Time PCR Instrument (Applied Biosystems™) using TaqMan™ OpenArray™ Real-Time PCR Master Mix as per the manufacturer’s instructions with the following protocol: 50°C for 2 min, 95°C for 2 min, 95°C for 1s and 60°C for 20s for 40 cycles. The ΔΔCt method was used for data analysis of RT-qPCR experiments.

Daw C.C., Ramachandran K., Enslow B.T., Maity S., Bursic B., Novello M.J., Rubannelsonkumar C.S., Mashal A.H., Ravichandran J., Bakewell T.M., Wang W., Li K., Madaris T.R., Shannon C.E., Norton L., Kandala S., Caplan J., Srikantan S., Stathopulos P.B., Reeves W.B, & Madesh M. (2020). Lactate Elicits ER-Mitochondrial Mg2+ Dynamics To Integrate Cellular Metabolism. Cell, 183(2), 474-489.e17.

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Cattle Gene Expression Profiling by qPCR

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The expressions of 18 genes (Table 3) were evaluated using real-time PCR. Three genes were used as endogenous controls to calculate the relative expressions of the other 15 candidate genes. The selection of reference and target genes was based on results observed in previous transcriptomics studies, where the administration of rbST and anabolic agents to cattle was evaluated [13 (link),14 (link),15 (link),16 (link),17 (link),31 (link)]. Gene-expression assays were carried out with a TaqMan^® OpenArray^® system (Applied BiosystemsTM, Thermo Fisher Scientific), involving a nanoliter high-throughput real-time PCR platform where 3072 reactions were performed simultaneously in the same OpenArray^® plate, and the primers and TaqMan^® probes were preloaded in the plates by the company. A plate design of 18 assays in triplicate, and 56 samples was chosen. Real-time PCR reactions were performed according to the TaqMan^® OpenArray^® protocol. Briefly, in a 384-well plate, 1.2 µL of each cDNA sample was mixed with 3.8 µL of TaqMan^® OpenArray^® Real-Time PCR Master Mix (Applied BiosystemsTM, Thermo Fisher Scientific). The PCR reaction mixtures were loaded automatically into the OpenArray^® plates using an OpenArray^® AccuFill™ System (Applied BiosystemsTM, Thermo Fisher Scientific). The following real-time PCR protocol was used: 40 cycles at 95 °C for 15 s, and 60 °C for 1 min.

Lamas A., Regal P., Vazquez B., Miranda J.M., Cepeda A, & Franco C.M. (2018). Tracing Recombinant Bovine Somatotropin Ab(Use) Through Gene Expression in Blood, Hair Follicles, and Milk Somatic Cells: A Matrix Comparison. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(7), 1708.

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Rat MBH Gene Expression Analysis

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1,000 ng of total RNA from rat MBH was reverse transcribed (RT) using the Omni RT Kit (Qiagen, Valencia, CA) in the presence of random hexamer primers (Invitrogen, Carlsbad, CA), as recommended by the manufacturer. The resulting complementary DNA (cDNA) was diluted four times with H₂O and mixed with 2 × TaqMan OpenArray Real-Time PCR Master Mix (Life Technologies, Grand Island, NY) at a ratio of 3.8:1.2 (PCR mix:cDNA). The mix was loaded into custom-made (12 × 224 probes) OpenArray plates (for target genes see Supplementary Data 5; for probe numbers and lengths of amplicon see Supplementary Data 9) using the QuantStudio OpenArray AccuFill platform and the PCR reactions were performed in a QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems, Foster City, CA). Experiments were performed two times.

Lomniczi A., Wright H., Castellano J.M., Matagne V., Toro C.A., Ramaswamy S., Plant T.M, & Ojeda S.R. (2015). Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression. Nature Communications, 6, 10195.

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Measuring Gene Expression Changes in Rat ARC

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We used the OpenArray-qPCR platform to measure changes in relative expression for 224 genes studied after perturbing the system by overexpressing EED in the ARC (rats injected on PND21 and euthanized on PND28; ARC-ME fragments collected after a 4 h incubation period to measure changes in pulsatile GnRH release). One thousand ng of total RNA from each ARC-ME fragment were reverse transcribed (RT) using the Omni RT Kit (Qiagen, Valencia, CA) in the presence of random hexamer primers (Invitrogen, Carlsbad, CA), as recommended by the manufacturer. The resulting cDNA was diluted 4 times with H2O and mixed with 2× TaqMan OpenArray Real Time PCR Master Mix (Life Technologies, Grand Island, NY) at a ratio of 3.8:1.2 (PCR mix:cDNA). The mix was loaded into custom made (12 × 224 probes) OpenArray plates (for target genes probe numbers and lengths of amplicon see^{20 (link)}) using the Quant Studio OpenArray AccuFill platform and the PCR reactions were performed in a QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems, Foster City, CA).

Wright H., Aylwin C.F., Toro C.A., Ojeda S.R, & Lomniczi A. (2021). Polycomb represses a gene network controlling puberty via modulation of histone demethylase Kdm6b expression. Scientific Reports, 11, 1996.

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Genetic Diagnostic Panel on QuantStudio 12K

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The genetic diagnostic panel was developed on the basis of QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The reaction mixture consisted of a DNA sample with 2 × TaqMan OpenArray Real-Time PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and was loaded onto the OpenArray plates using QuantStudio 12K Flex AccuFill system (Thermo Fisher Scientific, Waltham, MA, USA). The plates were coated with immersion liquid and loaded into QuantStudio 12K Flex Real-Time PCR System for amplification according to the manufacturer’s standard protocol. Data analysis was performed using the TaqMan Genotyper Software package, version 1.4.0 (Thermo Fisher Scientific, Waltham, MA, USA).

Kiseleva A., Klimushina M., Sotnikova E., Skirko O., Divashuk M., Kurilova O., Ershova A., Khlebus E., Zharikova A., Efimova I., Pokrovskaya M., Slominsky P.A., Shalnova S., Meshkov A, & Drapkina O. (2020). Cystic Fibrosis Polymorphic Variants in a Russian Population. Pharmacogenomics and Personalized Medicine, 13, 679-686.

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Quantifying PYCR1 and miR-150-5p Expression

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The total RNA from the tissues and cells was extracted using Trizol (Invitrogen, USA) and was reverse transcribed using a cDNA Reverse Transcription Kit (Liankebio, China). SYBR Premix Ex Taq (Takara, Japan) was used to detect PYCR1 expression. Tissue and cell miRNAs were extracted using an miRNA Isolation Kit (Thermo Fisher, USA) and reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher, USA). The TaqMan OpenArray Real-Time PCR Master Mix (Thermo Fisher, USA) was used to detect hsa-miR-150-5p expression. The expression of PYCR1 and hsa-miR-150-5p was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Uracil6 (U6), respectively, using the 2-^ΔΔCt method [18 (link)]. The primers used are listed in Table 1.Table 1.

Sequence of PCR primers used in this study

Gene name	Primer type	Sequence
miR-150-5p	Forward	5ʹ-TCGGCGTCTCCCAACCCTTGTAC-3ʹ
miR-150-5p	Reverse	5ʹ-GTCGTATCCAGTGCAGGGTCCGAGGT-3ʹ
CSE1L	Forward	5ʹ-TGACCAA-CACTCCAGTCGTG-3’
CSE1L	Reverse	5ʹ-GTCCAGCTTCACCTTGTCCA-3’
GAPDH	Forward	5ʹ-GGAGCGAGATCCCTCCAAAAT-3’
GAPDH	Reverse	5ʹ-GGCT-GTTGTCATACTTCTCATGG-3’
U6	Forward	5ʹ-CTCGCTTCGGCAGCACATATACT-3ʹ
	Reverse	5ʹ-ACGCTTCACGAATTTGCGTGTC-3ʹ

Li Z., Zhou X., Huang J., Xu Z., Xing C., Yang J, & Zhou X. (2021). MicroRNA hsa-miR-150-5p inhibits nasopharyngeal carcinogenesis by suppressing PYCR1 (pyrroline-5-carboxylate reductase 1). Bioengineered, 12(2), 9766-9778.

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