Total RNA were treated with DNAse1 and reverse transcribed to cDNA by Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, K1622) with oligo dT primers. Quantitative RT-PCR was performed using TaqMan probes and primer sets (Applied Biosystems) specific for CD36 (assay ID Mm00432398_m1), Dgat1 (Mm00515643_m1), Pparg (Mm00440940_m1), Cebpa (Mm00514283_s1), Dlk1 (Mm00494477_m1), and Sox9 (Mm00448840_m1). Ribosomal protein 20 (assay ID Mm02342828_g1) was used as normalization control for quantification by the ddCt method. PCR reactions were performed using 10 µl cDNA in PikoReal 96 real-time PCR system (Thermo Scientific). Quantification data were analyzed by two-tailed, homoscedastic t-tests based on the assumption that variances between the two sample data ranges are equal to type 2 Student’s t-test.
Taqman probes and primer sets
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan probes and primer sets are molecular biology tools used for quantitative real-time PCR (qRT-PCR) analysis. They are designed to detect and quantify specific DNA or RNA sequences with high specificity and sensitivity. The core function of TaqMan probes and primer sets is to facilitate the amplification and detection of target nucleic acid sequences in a qRT-PCR workflow.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Pikoreal 96 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Stepone software version 2, by Thermo Fisher Scientific (1 mentions)
Mm99999915 g1, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Roche (1 mentions)
Steponeplus, by Roche (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Bulge loop mirna qrt pcr primer set, by RiboBio (1 mentions)
Sybr fast universal qpcr kit, by Roche (1 mentions)
M mlv transcriptase, by Promega (1 mentions)
Reverse transcription system kit, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
8 protocols using taqman probes and primer sets
1
Quantitative RT-PCR Analysis of Adipocyte Genes
2
Real-Time PCR Analysis of RNA Expression
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Total RNA was isolated using RNeasy mini kit (Qiagen) and reverse transcribed to cDNA using an oligo (dT) with reverse transcriptase (Takara Bio), as previously described [74 (link)]. For real-time PCR, cDNA were amplified with the Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) or TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA). Data acquisition and analysis were performed with a Step One Real-Time PCR System using Step One Software, Version 2.1 (Applied Biosystems). The PCR products were quantified using Gapdh as the reference gene. The TaqMan probes and primer sets for detection of Tmem2 (Mm00459599_m1) and Gapdh (Mm99999915_g1) were purchased from Applied Biosystems. Primers for detection of Itgb1, Itgb3, Itga1 and Itga2 were shown in S3 Table .
3
Molecular Analysis of Circadian Genes
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Tissues used for molecular analysis were dissected immediately after decapitation at ZT12, frozen in liquid nitrogen, and stored at −80 °C until use. Tissue preparation and analysis were performed as previously described22 (link). We used pre-designed, gene-specific TaqMan probes and primer sets (Applied Biosystems, Foster City, CA) to assess the expression of the following genes: HDC (Mm00456104_m1) and CRH (Mm01293920_s1). Real-time RT-PCR was carried out using an Applied Biosystems StepOnePlus and TaqMan universal PCR Master Mix (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. For endogenous quantity control, we normalized values to those for the housekeeping gene β-actin (Mm00607939_s1).
4
Quantifying miRNA and mRNA Levels in ccRCC
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Total miRNA was isolated from cultured cells and surgically resected fresh ccRCC tissues using a mirVana miRNA Isolation Kit (Ambion, Austin, TX). The miRNA levels were assayed with Taqman probes and primer sets (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's instructions. Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific for miR-106b-5p are designed by RiboBio (Guangzhou, China). Extraction of total RNA and measurement of mRNA quantity were performed as described previously [32 ]. RNA was extracted from cells using TRIzol (Invitrogen) following protocols supplied by the manufacturer. First-strand cDNA was generated by MMLV transcriptase (Promega) using random primers. Real-time RT–PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad), and a Roche SYBR FAST Universal qPCR Kit (RocheMolecular Biochemicals) was used for gene detection. The remaining qPCR primers are listed in Supplementary Table 1 )
5
Temporal Cortex RNA Expression Analysis
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Total RNA was extracted from 50 mg of frozen tissue prepared from the superior temporal gyrus, as described in detail elsewhere.18 (link),19 (link),30 (link) Brain specimens from 109 controls samples with Caucasian ancestry were used. The messenger RNA (mRNA) level of DRD1 was measured by quantitative PCR using TaqMan probes and primer sets (Applied Biosystems, Foster City, CA, USA). TaqMan probe identification numbers are listed in Supplementary Table 1 . For relative quantification of mRNA expression, geometric means of the expression of three housekeeping genes (GUSB, PPIA, and RPLPO) were calculated using the standard curve method.
6
Quantification of mRNA and miRNA Levels
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Total RNA was extracted using TRIzol Reagent (Invitrogen). MiRNA levels were assayed using TaqMan ® probes and primer sets (Applied Biosystems, USA) according to the manufacturer's instructions. For mRNA analyses, the first-strand cDNA was generated using the Reverse Transcription System Kit from Promega with random primers used for reverse-transcription polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR (qRT-PCR) using a Power SYBR Green PCR Master Mix (Applied Biosystems) protocol in a StepOnePlus System (Applied Biosystems). The level of GAPDH mRNA was used as the internal normalization control. For precise quantification of gene copies per cell, Sox3 or reverse-transcribed miR-194 cDNA was used as standard templates to formulate standard curves with limiting dilution analysis. Then, exact copies of Sox3 and miR-194 per cell were calculated according to their molecular weight and cell counts. Primer sequences are presented in Table 2.
7
RNA Extraction and qPCR Analysis
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RNA was extracted using TRIzol (Invitrogen) per the manufacturer’s protocol. RNA was DNase treated and reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen), and gene expression was measured using a MX3000 qPCR machine (Stratagene). TaqMan primer and probes sets (Applied Biosystems) were used to measure gene expression. mGapdh was used as an endogenous reference gene, and relative gene expression was calculated using the ΔΔCT method.
8
TaqMan Assays for Murine Immunomodulatory Genes
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TaqMan primer and probes sets were purchased from Applied Biosystems: Ahr (Mm00478930_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cd274 (Mm00452054_m1), Ifnγ (Mm01168134_m1), Cyp1b1 (Mm00487229_m1), Cyp1a1 (Mm00487218_m1), and Gapdh (Mm99999915_g1). See SI Appendix for details.
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