qRT-PCR reactions were performed on LightCycler 1536 Multiwell plates (Roche, Basel, Switzerland). In each reaction, 23.5 ng of cDNA was mixed with LightCycler 1536 DNA Probes Master (Roche), and the appropriate TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA; CB1: Hs01038532_m1, CB2: Hs00275635_m1, ACTB: Hs99999903_m1). The reaction mixtures and samples were pipetted using an Echo Liquid Handler (Roche). The volume of the reaction mixture was 882.5 nL, and the volume of each sample was 117.5 nL. Each sample was applied to the plate in four replicates. Plates were amplified using a LightCycler 1536 instrument. The temperature and amplification time were set according to the protocol supplied with the TaqMan Gene Expression Assays, and 50 amplification cycles were performed. ACTB (coding for actin β) was amplified as a reference gene. Fluorescence signals and cycle threshold (CT) values were evaluated using the LightCycler 1536 Software, ver. 1.1. ΔCT values were calculated by normalization to ACTB.
Lightcycler 1536 dna probes master
Manufactured by Roche
Sourced in United States
The LightCycler 1536 DNA Probes Master is a real-time PCR (polymerase chain reaction) reagent designed for high-throughput nucleic acid amplification and detection. It contains the necessary components for performing real-time PCR assays, including DNA polymerase, dNTPs, and fluorescent probes.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rnalater stabilization solution, by Thermo Fisher Scientific (1 mentions)
Lc480 machine, by Roche (1 mentions)
Qbase, by CellCarta (1 mentions)
Lightcycler 480 high resolution melting dye, by Roche (1 mentions)
2 protocols using lightcycler 1536 dna probes master
1
Quantitative PCR Analysis of Cannabinoid Receptors
2
Transcriptional Analysis of Slc2a10 and EREs
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Skin and aortic tissue from 3 male and 3 female mice of each selected genotype were harvested at the age of 9 months and submerged in RNAlater™ Stabilization Solution (Thermo Fisher Scientific, cat. nr. AM7020) prior to the RNA extraction procedure, carried out with the RNeasy® Mini kit (Qiagen, cat. nr. 74106). Next, cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio-Rad, cat. nr. 1708891). RT-qPCR reactions were carried out in quadruple on a LC480 machine (Roche). The reaction mix consisted of cDNA, LightCycler® 1536 DNA Probes Master (Roche, cat. nr. 05502381001), LightCycler® 480 High Resolution Melting Dye (Roche, cat. nr. 04909640001) and specific primers amplifying the Slc2a10 transcript or Expressed Repeat Elements (EREs) (Supplementary Table 2) [28] ,
to which all reactions were normalized. Data analysis was carried out with qBase+ (Biogazelle). Values plotted correspond to the means of 3 male and 3 female biological replicates. Error bars shown represent 95% confidence intervals. Data were analyzed using Welch's ANOVA, followed by a Games-Howell post-hoc test.
to which all reactions were normalized. Data analysis was carried out with qBase+ (Biogazelle). Values plotted correspond to the means of 3 male and 3 female biological replicates. Error bars shown represent 95% confidence intervals. Data were analyzed using Welch's ANOVA, followed by a Games-Howell post-hoc test.
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