AMY1 copy numbers were measured in biobanked samples of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated by centrifuging fasting whole blood samples collected in BD Vacutainer® CPT™ cell preparation tubes with sodium citrate. The harvested PBMC pellet was resuspended in fetal bovine serum (FBS) with 10% dimethylsulphoxide (DMSO) and stored at −80 °C until analysis. Genomic DNA from PBMCs was isolated using the Maxwell 16 Cell LEV DNA purification kit (Promega, Fitchburg, WI, USA), and its purity and concentration was assessed using a NanoDrop One spectrophotometer (Thermofisher, Waltham, MA, USA). AMY1 gene copy numbers were estimated by duplex quantitative real-time PCR (2qPCR) on a Life Technologies QuantStudio™ 12K Real-Time PCR system, with QuantStudio™ software version 1.2.2, with a protocol adapted from Falchi et al. [9 (link)], and analysed with CopyCallerR software version 2.1 (Thermo Fisher Scientific, Waltham, MA, USA). Each sample reaction consisted of two TaqMan CNV assays (Life Technologies, Carlsbad, CA, USA); one specific for the target, AMY1 (Hs07226362_cn), and one specific for the reference gene (RNase P). Each sample was run in quadruplicate, and each run included an externally validated 14-copy control (NA18972, Coriell Cell Repositories, Camden, NJ, USA) along with a negative control.
Quantstudio 12k real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 12K Real-Time PCR System is a high-throughput real-time PCR instrument designed for gene expression analysis, genotyping, and other real-time PCR applications. The system features 12 independently controlled thermal blocks, allowing for up to 12 different experiments to be run simultaneously. The system is capable of detecting up to 6 fluorescent dyes and supports a wide range of sample volumes and plate formats.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Ribopure blood kit, by Thermo Fisher Scientific (2 mentions)
High capacity reverse transcriptase kit, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Taqman cnv assay, by Thermo Fisher Scientific (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Vacutainer cpt, by BD (1 mentions)
Na18972, by Coriell Institute for Medical Research (1 mentions)
Maxwell 16 cell lev dna purification kit, by Promega (1 mentions)
Copycaller v2, by Thermo Fisher Scientific (1 mentions)
Vacutainer blood collection tube, by BD (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Custom taqman gene expression array card, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
15 protocols using quantstudio 12k real time pcr system
1
Measurement of AMY1 Copy Numbers
2
Genetic Profiling of Blood Samples for Obesity
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Blood samples were collected from each participant using two vacutainer blood collection tubes (BD Vacutainer blood collection tubes) containing the anticoagulant (K2 EDTA) and polymer gel. The tubes were coded with an identifying number. Blood samples were centrifuged to separate plasma and serum, and the blood components were aliquoted into tubes and placed for −800°C storage.
DNA was extracted from the blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s protocol. The purified DNA was quantitated, and quality ascertained using a NanoDrop spectrophotometer. For SNPs genotyping, an SNP array using quantitative real-time PCR on a Life Technologies Quantstudio 12K Real Time PCR system based on the cycle relative threshold method was employed. The results were entered and analysed with Life Technologies Copy Caller V.2.1 software. The reactions carried out were based on a two-step assay, each with two primers and a Taqman probe, one specific to the target of SNP. Overweight individuals were defined as those with BMI between 25 and 30, while those with BMI higher than 30 were considered as obese according to WHO standards.1
DNA was extracted from the blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s protocol. The purified DNA was quantitated, and quality ascertained using a NanoDrop spectrophotometer. For SNPs genotyping, an SNP array using quantitative real-time PCR on a Life Technologies Quantstudio 12K Real Time PCR system based on the cycle relative threshold method was employed. The results were entered and analysed with Life Technologies Copy Caller V.2.1 software. The reactions carried out were based on a two-step assay, each with two primers and a Taqman probe, one specific to the target of SNP. Overweight individuals were defined as those with BMI between 25 and 30, while those with BMI higher than 30 were considered as obese according to WHO standards.1
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol (Life Technologies), followed by cDNA conversion with the SuperScript III First-Strand Synthesis System (Life Technologies). Quantitative real-time PCR was performed using a QuantStudio 12 K Real-Time PCR System (Life Technologies) with Power SYBR Green PCR Master Mix (Applied Biosystems). GAPDH was used as a normalization control.
4
Measuring ACE2 mRNA Expression in Caco-2 Cells
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Caco-2 cells were incubated with increasing concentrations of olmesartan/telmisartan (0, 1, 10, 50 µg/mL), captopril (0, 1, 5, 10 µg/mL), lisinopril (0, 0.005, 0.05, 0.5 µg/mL), or vehicle (DMSO) for 24 h, 48 h, or 72 h. The mRNA was extracted by RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA synthesis was performed using a First Strand cDNA-Synthesis kit (Thermo Scientific, Schwerte, Germany) including random hexamers. The expression levels of ACE2 and β-actin were determined using SYBR Select Mix (Applied Biosystems, Austin, TX, USA) with QuantStudio™ 12K Real-Time PCR System (Applied Biosystems, Austin, TX, USA). For human ACE2 mRNA detection, the forward primer 5′-AATGGGTCTTCAGTGCTCTC-3′ and reverse primer 5′-GAGCCTCTCATTGTAGTC-3′ and for human β-actin the forward primer 5′-CCAACCGCGAGAAGATGA-3′ and the reverse primer 5′-CCAGAGGCGTACAGGGATAG-3′ were used. Relative mRNA expression was determined using the comparative CT (cycle threshold) method, normalizing relative values to the expression level of human β-actin.
5
Quantitative RT-PCR for Gene Expression
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Total RNA from cells was isolated with RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. A total of 100 ng of RNA was reverse-transcribed to cDNA with High-Capacity cDNA Reverse Transcription Kit (Life Technologies). For qPCR, TaqMan gene expression assays were used on a QuantStudio 12K Real-Time PCR system (Applied Biosystems). FAM-conjugated TaqMan probes for Hhip, Ifnγ, Il18, Il12a, Il12b, Gapdh, Cd8a, and Tbp (mouse) and HHIP, IL18, and GAPDH (human) (IDT) were used with TaqMan Gene Expression Master Mix (Applied Biosystems). Gene expression values were normalized to GAPDH for isolated fibroblasts and MRC5 cell lines and to Cd8a and Tbp for isolated CD8+ T lymphocytes and macrophages. Expression levels of target genes were calculated based on the 2-ΔΔCt method. For undetermined Ct values, Ct value of 40 was used to calculate relative expression level.
6
Customized TaqMan Low-Density Array for Gene Expression Analysis
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For the training set, customized TaqMan Low-Density Array (TLDA) 384-well microfluidic cards with inventoried predesigned assays were designed using the Custom TaqMan Gene Expression Array Card service from Life Technologies, Carlsbad, CA (format 96a). For the validation set, 96-well fast plates were used to carry out the reactions using specific predesigned TaqMan inventoried individual assays (Thermo Scientific, Waltham, MA). The preamplified cDNAs were mixed with 2 × TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA), and TLDA cards or 96-well plates analyzed the QuantStudio 12K Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Samples were heated at 50°C for 2 minutes and 95°C for 10 minutes and then subjected to 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. The Ct (cycle threshold) of each gene was obtained by setting the fixed threshold at 0.05.
7
Quantifying Antioxidant Gene Expression
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Total RNA was extracted from whole blood using a Ribopure Blood Kit (Ambion, Thermo Scientific, Austin, TX, USA) and final concentration and purity were measured spectrometrically in a NanoDrop ND 1000 spectrophotometer (NanoDrop ND 1000, Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized by reverse trancriptase PCR using a High Capacity Reverse Transcriptase kit (Applied Biosystems, Thermo Scientific, Fostercity, CA, USA). Analysis of gene expression was performed by real-time quantitative PCR (qPCR) in the QuantStudio 12K Real-Time PCR System using Taqman Gene Expression Assays for GPX1, SELENOP, SELENOS and SELENOF (Applied Biosystems, Thermo Scientific, Fostercity, CA, USA). Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA was used as a reference gene. Relative gene expression was calculated based on the 2−ΔΔCq method [25 (link)].
8
Quantitative Gene Expression Analysis of FFPE Breast Cancer Samples
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Custom designed 384-well TaqMan array cards (ThermoFisher) were used to measure gene expression in FFPE breast cancer samples. Primer sequences were designed by ThermoFisher. Array cards were loaded with 100 μL of 1:1 mix of cDNA and TaqMan Fast Advanced Master Mix (Applied Biosystems) and run using a QuantStudio12K Real-Time PCR system (Applied Biosystems), as per the manufacturer’s instructions. The expression of each gene was measured in duplicate.
9
Quantitative PCR Analysis of Antibacterial and Inflammatory Responses
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The material from 2 to 3 monolayers, which underwent the same stimulus, was pooled prior to RNA extraction. Briefly, total RNA was isolated with the Reliaprep RNA Cell Miniprep System (Promega) following the manufacturer's instructions. For reverse‐transcription of RNA into complementary DNA synthesis (cDNA), RT2 First Strand Kit (Qiagen) was used. Following manufacturer's instructions, both cDNA and RT2 SYBR® Green qPCR Mastermix (Qiagen) were used on a custom RT2 Profiler PCR array (Qiagen) targeting 86 genes related to antibacterial and inflammatory response, and epithelial barrier integrity. PCR arrays were analyzed in a QuantStudio 12K Real‐Time PCR System (Applied Biosystems™, Life Technologies). CT values were removed if undetermined; CT > 36 was adjusted to CT = 38. Genes were excluded from analysis if >60% of samples had missing or very high CT values (n = 11). Gene expression was calculated as 2−∆CT (2−(CT Target gene−CT Housekeeping gene) and the average of the housekeeping genes ACTB, B2M, GAPDH, HPRT1 and RPLP0 was used for normalization. A complete list of the genes targeted in the custom array is shown in Data S1 .
10
Apoptosis Gene Expression Profiling
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The expression of an extensive number of apoptosis associated genes was determined using a TaqMan Array Human Apoptosis, Fast 96-well Plate (Part No. 4418717, Applied Biosystems, Foster City, CA, USA). The TaqMan Array Plate contained 92 assays for apoptosis associated genes and 4 assays for endogenous control genes. The RT-qPCR amplification was performed on a Quantstudio 12K Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in a 10 μL final volume, containing TaqMan Fast Advanced Master Mix (2x) (Applied Biosystems, Foster City, CA, USA) and cDNA samples from cells treated with 10 μg/mL BG venom, 10 μg/mL DA venom or untreated cells (negative control). Gene expression was analyzed using Expression Suite Software (version 1.1., Thermo Fisher Scientific, Waltham, MA, USA).
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