Total RNA isolated from ventral midbrain samples served as a template for DNA synthesis using Super-Script III first-strand synthesis kit (Invitrogen). For genomic DNA contamination control, samples with no added reverse transcriptase enzyme were included. Quantitative PCR was performed with a CFX96 Real-Time System (Bio-Rad) using TagMan Gene Expression Assays (Life Technologies) according to the manufacturer's instructions. The mRNA levels of Hprt1 were measured to control for the equal amount of input cDNA. The following probes were used for detection of En1, Foxa1, Foxa2, Hprt1, Lmx1b, Nr4a2, Pitx3, Th, and Ucp2: Mm00438709_m1, Mm00484713_m1, Mm00839704_mH, Mm01545399_m1, Mm00440209_m1, Mm00443056_m1, Mm01194166_g1, Mm00447557_m1, and Mm00495907_g1, respectively.
Tagman gene expression assays
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Gene Expression Assays are a set of pre-designed and pre-optimized real-time PCR assays for measuring gene expression. They provide a reliable and consistent method for quantifying mRNA levels of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Cfx1000, by Bio-Rad (1 mentions)
Faststart essential dna probes master, by Roche (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
96 well optical reaction plate, by Thermo Fisher Scientific (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
7500 system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix reagent, by Thermo Fisher Scientific (1 mentions)
Rt pcr kit, by Takara Bio (1 mentions)
7900ht fast system, by Thermo Fisher Scientific (1 mentions)
Taqman fast cells to ct, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tagman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
7 protocols using tagman gene expression assays
1
Transcriptional Profiling of Midbrain Neurons
2
Quantitative Gene Expression Analysis
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RNA was prepared using the RNeasy kit (Qiagen) and was reverse transcribed using Superscript II (Life Technologies) according to standard protocols. RNA equivalent cDNA (5 ng) was used to perform real-time PCR using predesigned tag-man gene expression assays (Life Technologies) using a BioRad CFX1000. Gene expression levels of mouse Gch1, Nos1, Nos2, and Nos3 were normalized to the housekeeping gene GAPDH using the ΔCt method.
3
PPAR-γ Expression Analysis in Prostate Cancer
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Complimentary DNA was synthesized by reverse transcription using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany). 1 µg of total RNA were reverse transcribed with a combination of anchored-oligo (dT) and random hexamer primers, according to the manufacturers' instructions. To assess PPAR-γ (Hs01115513) and GAPDH (Hs99999905) gene expression, we used pre-designed quantitative real-time TagMan Gene Expression Assays (Applied Biosystems, Foster city, CA, USA). Quantitative real-time PCR reactions were performed in final volume of 20 µL using 10 ng cDNA/well and 10 µL FastStart Essential DNA Probes Master (Roche Diagnostics) according to the manufacturers' instructions. Thermal cycling conditions on the LightCycler Nano System were the following: Enzyme activation: 95℃ for 10 min, 45 cycles of amplification: 95℃ for 10 sec, 60℃ for 30 sec. Each quantitative real-time PCR analysis was performed in triplicate. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control gene. The normalized amount of each target mRNA present in 20 PCAs cases was calculated by benign prostate tissue cases.
4
RNA Extraction and qPCR Analysis
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Example 6
Cultured cells were treated in the same way as described for the immunoblot experiments. After 12 h, total RNA was extracted using a commercial kit (RNeasy Mini Kit, Qiagen). For mice tissues, total RNA was extracted using TRIzol® reagent (Invitrogen) following the standard manufacturer's protocol.
cDNA synthesis and qPCR experiments for both cultured cells and mice tissues were performed following the same procedure. 2 μg of each RNA sample was reverse-transcribed into cDNA, which served as a template for TagMan® gene expression assays (Applied Biosystems). For each qPCR run, a total reaction volume of 25 μl was prepared consisting of 12.5 μl of TaqMan 2× universal master mix, 1.25 μl of a 20× TaqMan gene expression assay probe, 1 μl of cDNA and 10.25 μl of RNase-free sterile water. The reactions were dispensed into 96-well optical reaction plates (Applied Biosystems) and detected in an Applied Biosystems® 7300 Real-Time PCR System. The thermocycler program consisted of 2 min at 50° C., 10 min at 95° C., and 40 cycles of 95° C. for 15 s and 60° C. for 1 min. For normalization, GAPDH expression was used as an internal control for human cells (IMR-32) and β-actin for mice tissue samples.
5
Quantitative PCR Analysis of TAK1
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qPCR was performed using the oligonucleotides/TagMan probes provided for TagMan Gene Expression Assays (Applied Biosystems, Foster City, CA) (TAK1, Assay ID: HS01105682_m1; 18S, Catalog Number: 4310893E) in an ABI 7500 system according to the manufacture's instruction. Expression levels of target genes were quantified against endogenous 18S level using the comparative CT method. The 7500 system SDS Software was used for data analysis.
6
Quantitative Analysis of LRP2 mRNA Expression
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Total RNA was extracted from each tissue or cell using a TRIzol reagent (Invitrogen, CA) and subjected to quantitative real-time PCR as described56 (link). Single stranded cDNA from the total RNA was synthesized with a RT-PCR kit (Clontech, Mountain View, CA) according to the kit’s instructions. Quantitative RT-PCR was performed with an Applied Biosystems 7900HT Fast System using either a SYBR Green PCR Mastermix reagent (Applied Biosystems, Foster City, CA) or TagMan® Gene Expression Assays (Applied Biosystems). Relative mRNA expression levels were determined using the 2−ΔΔCT method normalized to 36B4. For LRP2 mRNA in human muscle, the forward primer 5ʹ-CCA GCA AGG AAC CAG AGA ACA-3ʹ and the reverse primer 5ʹ-AGG CAG AGC AAA GCA GAG ATG-3ʹ were used. For LRP2 mRNA in C2C12 cells, the forward primer 5ʹ-GAG TGC ATC CTT CGT GCC TAT-3ʹ and the reverse primer 5ʹ-CAG CCA TCC TCA TCA CCA GAA-3ʹ were used. For LRP2 mRNA in mouse muscle, the commercial primers for LRP2 (Mm01328171_m) and 18 s (Mm0427787_s1) (Applied Biosystems) were used.
7
Quantitative Analysis of Chondrogenic Gene Expression
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Total RNA was extracted using TaqMan ® Fast Cells-to-CT™, (Ambion, Life Technologies) according to the manufacturer's instructions. In brief, each sample was lysed and reverse transcriptase polymerase chain reaction (PCR) was performed on cell lysate, which was transcribed into cDNA using TaqMan ® Fast Cells-to-Ct™ (Ambion). Quantitative real-time RT-PCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using TagMan ® Fast Universal PCR Master Mix (Applied Biosystems) and TagMan ® Gene Expression Assays (Applied Biosystems) with the following assay: sex-determining region Y box 9 (SOX9) Hs00165814_m1, Collagen type 2 alpha 1 (Collagen II); Hs00264051_m1, aggrecan (Aggrecan); Hs00153936_m1, and Collagen type 1 alpha 1 (Collagen I); Hs00164000_m1. Standard enzyme and cycling conditions for the 7500 Fast System were used. Each biological sample was run in technical duplicates for each gene. Data analysis was performed using 7500 Fast System Sequence Detection Software version 3.1 (Applied Biosystems). Target gene expression was normalized to the housekeeping genes (HKG) Beta-2-microglobulin (B2M) Hs99999907_m1 and ribosomal protein L13a (RPL13a) Hs03043885_g1 based on BestKeeper values (34) , (37) .
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