RNA was isolated from cells in culture using the RNeasy Plus Mini Kit (Qiagen, Manchester, UK) according to manufacturer's instructions. Reverse transcription was carried out using 1 μg RNA per reaction using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Loughborough, UK) according to kit instructions. The resulting cDNA was diluted one in 10 for RT‐quantitative PCR (qPCR) analysis. cDNA panels of non‐neoplastic tissue were purchased from Clontech (Saint‐Germain‐en‐Laye, France). TaqMan probes were used as stated in each experiment according to the manufacturer's instruction. Human assays were obtained from Thermo Fisher (Loughborough, UK): LINC00261 (Hs03679073_m1), LINC00491 (NEAR3) (Hs01374494), LOC101929331 (NEAR5) (Hs04406674_m1), LOC100507175 (NEAR8) (Hs01388460_m1), LINC01612 (NEAR16) (Hs04407222_m1), CTD‐2151A2.1 (NEAR18) (Hs04404647), LINC00616 (NEAR21) (Hs04232282_m1), HPRT1 (Hs02800695), FOXA2 (Hs00232764_m1), and CBX2 (hs01034268_m1). Murine assays were also obtained from Thermo Fisher 9030622O22‐Rik (Mm01335331_m1), and HPRT1 (Mm03024075_m1).
Hprt1
Manufactured by Thermo Fisher Scientific
Sourced in United States
HPRT1 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a key enzyme involved in the purine salvage pathway, which is responsible for the recycling of purine nucleotides. The HPRT1 gene encodes the hypoxanthine-guanine phosphoribosyltransferase enzyme, which plays a crucial role in maintaining cellular levels of guanine and adenine nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Thermo Fisher Scientific (6 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (6 mentions)
Rneasy plus mini kit, by Qiagen (5 mentions)
Taqman assay, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (3 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Total rna mini kit, by A&A Biotechnology (2 mentions)
T100 thermal cycler, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Nucleospin rna kit, by Macherey-Nagel (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
H3k27m, by Roche (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
H3k27me3, by Merck Group (2 mentions)
47 protocols using hprt1
1
RNA Isolation and RT-qPCR Analysis of LncRNAs
2
Comprehensive Gene Expression Analysis
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Total RNA was purified using the RNeasy Plus Mini Kit (Qiagen, Copenhagen, Denmark). The RNA concentration was measured by the Qubit RNA HS kit and the Qubit 4 Fluorometer (ThermoFisher Scientific) and stored at −80 °C until use. Two μg total RNA was reverse transcribed using the RevertAid Minus First strand cDNA synthesis kit (ThermoFisher Scientific) using oligo(dT) primers, following the manufacturer’s instructions. qPCR was performed with TaqMan Fast Advanced Master Mix TaqMan assays for SOX2 (Hs01053049_s1), NANOG (Hs04260366_g1), POU5F1 (Hs0099632_g1), CD44 (Hs01075864_m1), PROM1 (Hs01009250_m1), BAD (Hs00188930_m1), BAK1 (Hs00832876_g1), BAX (Hs00180269_m1), BCL2 (Hs00608023_m1), TP53 (Hs01034249_m1), CDKN1A (Hs00355782_m1), HPRT1 (Hs02800695_m1), and GAPDH (Hs99999905_m1), all from ThermoFisher Scientific. Ten ng cDNA was used per reaction, as recommended by the manufacturer’s protocols. The qPCR was performed in a QuantStudio 3 (ThermoFisher Scientific) for 2 min at 50 °C, 2 min at 95 °C, followed by 40 cycles at 95 °C for 1 sec and 60 °C for 20 s. Expression levels were normalized to HPRT1 and GAPDH, and relative expression levels were calculated using the Pfaffl-method [54 (link)].
3
Gene Expression Analysis in Neurodegenerative Disorders
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cDNA was synthesized using Taqman™ III Universal PCR mastermix (4305719; Applied Biosystems, Life Technologies). The reactions in 96‐well plates were performed with cDNA Thermal cycler (Eppendorf Mastercycler Pro) under 10 min at 25°C, 120 min at 37°C, 5 min at 85°C, and ∞ at 4°C. TaqMan probes used were HPRT1 (Hs02800695_m1; Thermo Fisher) as a housekeeping gene, SNCA (Hs00240906_m1; Thermo Fisher), LAG3 (Hs00958444_g1; Thermo Fisher), and TLR2 (Hs00152932_m1).
qPCR reactions were run with the following cycling parameters: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min by real‐time PCR instrument (QuantStudio 7; Applied Biosystems). The means of the groups were calculated and compared using QuantStudio™ Real‐Time PCR Software (Version 1.3) and ΔΔCt (comparative Ct) method.
qPCR reactions were run with the following cycling parameters: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min by real‐time PCR instrument (QuantStudio 7; Applied Biosystems). The means of the groups were calculated and compared using QuantStudio™ Real‐Time PCR Software (Version 1.3) and ΔΔCt (comparative Ct) method.
4
Quantifying Mouse Hepatocyte ANGPTL4 mRNA
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About 15,000 primary mouse hepatocytes (Lonza) per well were seeded in BioCoat Collagen I 96-well flat-bottom plates (catalog no.: A11428-03; Thermo Fisher Scientific) in 100 μl Hepatocyte Plating Medium (catalog no.: MP250; Lonza). Supernatant was removed 4–6 h after seeding, and ASOs were added at indicated concentrations (5,000, 1,000, 200, 40, 8, and 1.6 nM) diluted in 100 μl Maintenance Medium (catalog no.: MM250; Lonza). Cells were cultured for 3 days at 37°C. Afterward, the cells were lysed, and mRNA levels were measured according to the manufacturer's instructions, using the QuantiGene Singleplex Gene Expression Assay (catalog no.: QS0011; Thermo Fisher Scientific) and the following probesets: murine ANGPTL4 (catalog no.: SB-16744-02; Thermo Fisher Scientific) and HPRT1 (catalog no.: SB-15463; Thermo Fisher Scientific). Values were normalized to the housekeeping gene HPRT1. IC50 values were calculated using Prism 6 (GraphPad Software, Inc). Data are represented as the mean of triplicate wells ± SD relative to untreated cells (set as 1).
5
Quantitative RT-PCR for SARS-CoV-2 Detection
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Total RNA was extracted from lung homogenates using QIAamp Viral RNA (Qiagen®), according to manufacturer’s instructions. Quantitative RT-PCR was performed using GoTaq® 1-Step RT-qPCR System (Promega) in a StepOne™ Real-Time PCR System (Thermo Fisher Scientific). Amplifications were carried out in 15 µL reaction mixtures containing 2× reaction mix buffer, 50 µM of each primer, 10 µM of the probe, and 5 µL of RNA template. Primers, probes, and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect the SARS-CoV-2 (CDC 2020). Amplification of the housekeeping gene HPRT1 (Mm03024075_m1, Thermo Fisher Scientific) was used as a reference for the number of cells used. The Ct values for this target were compared to those obtained with different cell quantities (107 to 102), for calibration.
6
Quantitative Analysis of Thyroid Receptor Genes
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Total RNA was extracted from the frontal cortex and hippocampus using a Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland), after which the RNA concentrations in the samples were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Conversion into cDNA was performed by a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States) using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, United States). Quantitative real-time PCR was performed using TaqMan probes and primers for the thra, thrb, rxra, rxrb, dio2, dio3, and thrsp genes (Thermo Fisher Scientific, Waltham, MA, United States) and the FastStart Universal Probe Master (Rox) kit (Roche, Basel, Switzerland) using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, United States). The following thermal cycling conditions were used: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The Ct values for each sample were measured in the exponential phase of the PCR, and the ΔΔCt method was used for data analysis. hprt1 (Thermo Fisher Scientific, Waltham, MA, United States) was used as the reference gene.
7
Quantifying Immune Gene Expression
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Total RNA was extracted from cells (1–2 × 106 cells per sample) with an RNeasy kit (Qiagen, Germantown, MD, USA) and cDNA was generated using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed on a Light Cycler 480 II (Roche, Indianapolis, IN, USA). Primers for IFIT1 (Hs03027069), IFI44 (Hs00197427), RAGE (Hs00542584), FcRIIa (Hs01013401), MX1 (Hs00182073,), HPRT1 (Hs99999909), and Polr2a (Hs00172187) were purchased from Thermo Scientific. The genes of interest were normalized to the expression of the house keeping gene (Polr2a) and were compared to a control condition with no treatment. The relative induction was calculated by 2−ΔΔCt.
8
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from about 106 cells from each condition by using the NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany; Diomede et al., 2018a (link)). RNA quantity and quality were assessed by Qubit 2.0 (ThermoFisher Scientific, Whaltam, MA, United States; Diomede et al., 2018b (link)).
1 μg of total purified RNA from each sample was reverse transcribed using the High Capacity RNA-to-cDNA Kit (ThermoFisher Scientific). qRT-PCR was performed in a total volume of 20 μL containing 2× Maxima SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific), 3 μL of cDNA and 0.3 μM of each primer. GAPDH and HPRT1 were used as housekeeping genes (ThermoFisher Scientific, Waltham, MA, United States; Gugliandolo et al., 2018 (link)). Real time amplification conditions were 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. A final melting dissociation curve was run to assess primers specificity. Each sample was run in triplicate. Specific primers pairs (IDT, Skokie, IL, United States) employed are reported inTable 1 The ΔΔCt method and the two-tailed t-test were employed to assess the relative gene expression, considering data significant when p < 0.05.
1 μg of total purified RNA from each sample was reverse transcribed using the High Capacity RNA-to-cDNA Kit (ThermoFisher Scientific). qRT-PCR was performed in a total volume of 20 μL containing 2× Maxima SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific), 3 μL of cDNA and 0.3 μM of each primer. GAPDH and HPRT1 were used as housekeeping genes (ThermoFisher Scientific, Waltham, MA, United States; Gugliandolo et al., 2018 (link)). Real time amplification conditions were 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. A final melting dissociation curve was run to assess primers specificity. Each sample was run in triplicate. Specific primers pairs (IDT, Skokie, IL, United States) employed are reported in
9
Quantification of Inflammatory Cytokines
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Extraction of total RNA was performed using Isolate II RNA mini kit (Bioline, London, UK). The RNA concentration and purity were measured using NanoDrop One C spectrophotometer (Nanodrop Technologies, Anaheim, USA). The resulting RNA concentrations were standardized using the lowest sample concentration and cDNA was made using the Tetro Reverse Transcriptase (Bioline). Quantitative PCR (qPCR) was performed using the QuantStudio 5 Real-Time PCR System (Thermo Fischer Scientific) with predeveloped TaqMan probe/primer combinations for human TNF (HS00174128), IL-6 (Hs00174131), IL-10 (Hs00961622) and internal control hypoxanthine phosphoribosyl transferase 1 (HPRT1) (4325801, Thermo Fischer Scientific). Threshold cycle numbers were transformed to cycle threshold values, and the results were plotted using GraphPad Prism version 9.5.
10
Quantitative Analysis of ALDH1A1 mRNA
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Real time quantitative RT-PCR was carried out with TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific) using the Viaa7 Flex Real-Time PCR System (Thermo Fisher Scientific) in 10 μl reaction volumes using 384 well plates. Taqman assays were used for ALDH1A1 (Thermo Fisher Scientific) and HPRT1 (Thermo Fisher Scientific). All reactions were carried out in triplicate and fold-change expression values were averaged. Relative difference in the gene expression was normalized to expression levels of a housekeeping gene, HPRT1. The average %CV (for individuals' triplicate samples) for ALDH1A1 mRNA expression analyzed by real time qPCR was 0.50 (range 0.05–2.0, based on Ct values).
For examining the effects of chronic TNF on ALDH1A1 mRNA expression, ME-SFCs (p0) (n = 19 control subjects) were treated with vehicle or TNF (10 ng/ml) on days 1 and 3. On day 7, ME-SFCs were lifted after a brief trypsinization and plated for assessment of ALDH1A1 mRNA expression, as described above.
For examining the effects of chronic TNF on ALDH1A1 mRNA expression, ME-SFCs (p0) (n = 19 control subjects) were treated with vehicle or TNF (10 ng/ml) on days 1 and 3. On day 7, ME-SFCs were lifted after a brief trypsinization and plated for assessment of ALDH1A1 mRNA expression, as described above.
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