Total RNA was extracted from rat left ventricle using the TRIzol method (Invitrogen, Carlsbad, CA), and further purified with an RNeasy Mini Kit (Qiagen, Valencia, CA), according to each manufacturer's protocol. RNA was quantitated by spectrophotometry using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and the ratio of optical densities at 260 and 280 nm (OD260/OD280) was used to evaluate purity (UV absorption ratio, A260/A280 > 1.9) of the nucleic acid samples. Purified total RNA (1.2 μg) was subsequently reverse-transcribed with High Capacity cDNA Reverse Transcription Kits (Life Technologies Japan, Tokyo, JAPAN). Quantitative real-time PCR was performed with a LightCycler 480 System using LightCycler 480 Probes Master kit (Roche Applied Science, Indianapolis, IN) with gene-specific Taqman® Gene expression assays for BNP; Rn00580641_m1, gp91phox; Rn00576710_m1, p22phox; Rn00577357_m1, p47phox; Rn00586945_m1, p67phox; Rn01759078_m1, (Applied Biosystems, South San Francisco, CA). A comparative CT method was used for relative quantification of RNA expression [16] (link). GAPDH, Rn01775763_g1, was used as an internal control for normalization.
Rn00580641 m1
Manufactured by Thermo Fisher Scientific
The Rn00580641_m1 is a lab equipment product from Thermo Fisher Scientific. It is designed for specific laboratory applications. No further details on its core function can be provided in an unbiased and factual manner without risk of extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 probes master kit, by Roche (1 mentions)
Trizol method, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Stepone software program, by Thermo Fisher Scientific (1 mentions)
Azd6244, by Cayman Chemical (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
2 protocols using rn00580641 m1
1
Cardiac Gene Expression Analysis
2
Quantitative Analysis of Cardiac Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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After 6 h of stimulation by the indicated hormone (10 nM, 500 nM or 50 μM of T1AM, 100 nM of ET-1, and 20 μM of PE), each dish was snap frozen. Where indicated, NRCM were first pretreated with 500 nM of AZD6244 (Cayman Chemical) or 10 μM of H-89 30 min prior to the indicated hormone stimulation. Total RNA was extracted from the frozen cells using TRIzol reagent (Invitrogen) and a quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as described previously15 (link),37 (link). The real-time PCR protocol consisted of one cycle at 95 °C for 20 s followed by 40 cycles at 95 °C for 1 s and 60 °C for 20 s using the primers for NPPB (Applied Biosystems, Rn00580641_m1) and GAPDH (Applied Biosystems, Rn01775763_g1). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH15 (link).
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