RNA expression assay was performed for Slc12a2 (Rn00582505_m1) and Glpdh (Rn01775763_g1) genes. High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, USA) was used for reverse transcription reaction in 20 μl reaction volume containing 50 ng of total RNA incubated at 25°C for 10 min, transcripted at 37°C for 120 min, and terminated by heating at 85°C for 5 min using Biometra TAdvanced thermocycler (Analytik Jena AG, Germany). The synthesized cDNA was stored at 4°C until use or at -20°C for longer time. The Real-time PCR was run in triplicate with 4 μl of cDNA template in a 20 μl reaction volume (10 μl of TaqMan Universal Master Mix II, no UNG (Applied Biosystems, USA), 1 μl of TaqMan Gene Expression Assay 20x (Applied Biosystems, USA), and 5 μl of Nuclease-Free Water (Invitrogen, USA) with the program running at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. the reaction was performed using an Applied Biosystems 7900 Fast Real-Time PCR System (Applied Biosystems, USA).
Taqman gene expression assay 20x
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan Gene Expression Assay 20x is a pre-designed and pre-optimized reagent for quantifying gene expression levels using real-time PCR technology. It provides a standardized and reliable solution for accurate and sensitive measurement of target gene transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Applied biosystems 7900 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Nuclease free water, by Thermo Fisher Scientific (2 mentions)
Biometra tadvanced thermocycler, by Analytik Jena (1 mentions)
Fast universal qpcr mastermix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Optical caps, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix 2x, by Thermo Fisher Scientific (1 mentions)
Optical fast 96 well plate, by Thermo Fisher Scientific (1 mentions)
5 protocols using taqman gene expression assay 20x
1
RT-qPCR Assay for Slc12a2 and Glpdh
2
Endogenous Control qPCR Assay
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Gene expression level for endogenous controls was determined using pre-validated Taqman Gene Expression Assays (Applied Biosystems). qPCR reactions for 384 well plate formats were performed using 2 μl of cDNA samples (5-25ng), 5μl of the Fast Universal qPCR MasterMix (Applied Biosystems), 0.5 μl of the TaqMan Gene Expression Assay (20x) and 2.5 μl of water in a total volume of 10 μl. The following assays were used as endogenous controls: HPRT1 (Rn01527840) and b-Actin (Rn00667869).
3
Cardiac Hypertrophy Gene Expression Analysis
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Total RNA from control and hypertrophic hiPSC-CM was extracted using the RNeasy mini kit (Qiagen, 74104) according to the manufacturer's instructions. Reverse transcription of extracted RNA was performed using the iScript cDNA synthesis kit (Bio-Rad, 1708891). Subsequent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted with 20 µl reaction mixture composed of 2 µl cDNA plus 7 µl RNase-free water, 1 µl TaqMan Gene Expression Assay (20X) (NPPB Hs00173590_m1; NPPA Hs00383230_g1; MYH6 Hs01101425_m1; MYH7 Hs01110632_m1) and 10 µl TaqMan Universal PCR Master Mix (Applied Biosystems, 4304437). Amplification was conducted in QuantStudio 5 with thermal cycling parameters as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Amplification curves were generated automatically by the real-time PCR system. Relative quantification compared target gene expression between control and hypertrophic groups using the comparative ΔCT method. GAPDH were used as internal control.
4
Quantitative Real-Time PCR Protocol
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A total volume of 20 µL containing 1 µL TaqMan® Gene Expression Assay 20 x (Table 3 ; Applied Biosystems), 10 µL TaqMan® Fast Universal PCR Master Mix 2 x (Applied Biosystems), 1 µL cDNA template and 8 µL H2O (DEPC-treated DI water, Sigma, St. Louis, MO, USA) was applied per probe on an Optical Fast 96-well plate (Applied Biosystems) and covered by optical caps (Applied Biosystems). Using Taqman 7500 Fast (Applied Biosystems), PCR assays were performed. At 95 °C for 20 s on hold, enzymes were activated followed by 40 cycles of qPCR denaturing at 95 °C for 3 s and annealing at 60 °C for 30 s. The Relative Quantification (RQ) = 2−∆∆Ct method, which is defined as the first fluorescent signal reaching statistical significance, was applied for the results. ∆Ct values were calculated by normalizing to β-actin, which was used as an endogenous control.
5
Quantification of RNA Expression Levels
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RNA expression assays were performed for Slc5a8 (Rn01503812_m1),
Slc12a2 (Rn00582505_m1), and Gapdh(Rn01775763_g1) genes. According to the manufacturer’s instruction, the reverse
transcription was performed with High-Capacity cDNA Reverse Transcription Kit
with RNase Inhibitor (Applied Biosystems, USA) in 20 μL reaction volume
containing 50 ng RNA using Biometra TAdvanced thermal cycler (Analytik Jena AG,
Germany). The synthesized copy DNA (cDNA) was stored at 4°C until use or at
−20°C for a longer time. Real-time polymerase chain reaction (PCR) was performed
using an Applied Biosystems 7900 Fast Real-Time PCR System (Applied Biosystems,
USA). The reactions were run in triplicate with 4 μL of cDNA template in a 20 μL
reaction volume (10 μL of TaqMan Universal Master Mix II, no UNG (Applied
Biosystems, USA), 1 μL of TaqMan Gene Expression Assay 20x (Applied Biosystems,
USA), 5 μL of nuclease-free water (Invitrogen, USA) with the program running at
95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The
amplification efficiency was about 100%. The amplicon length of TaqMan assays
was 60 bp for the Slc5a8 gene, Slc12a2—67 bp,
and Gapdh—174 bp.
Slc12a2 (Rn00582505_m1), and Gapdh(Rn01775763_g1) genes. According to the manufacturer’s instruction, the reverse
transcription was performed with High-Capacity cDNA Reverse Transcription Kit
with RNase Inhibitor (Applied Biosystems, USA) in 20 μL reaction volume
containing 50 ng RNA using Biometra TAdvanced thermal cycler (Analytik Jena AG,
Germany). The synthesized copy DNA (cDNA) was stored at 4°C until use or at
−20°C for a longer time. Real-time polymerase chain reaction (PCR) was performed
using an Applied Biosystems 7900 Fast Real-Time PCR System (Applied Biosystems,
USA). The reactions were run in triplicate with 4 μL of cDNA template in a 20 μL
reaction volume (10 μL of TaqMan Universal Master Mix II, no UNG (Applied
Biosystems, USA), 1 μL of TaqMan Gene Expression Assay 20x (Applied Biosystems,
USA), 5 μL of nuclease-free water (Invitrogen, USA) with the program running at
95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The
amplification efficiency was about 100%. The amplicon length of TaqMan assays
was 60 bp for the Slc5a8 gene, Slc12a2—67 bp,
and Gapdh—174 bp.
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