Total RNA extraction from different samples was done using the method previously described [41 (link)] and quantified using a UV spectrophotometer. The RT-PCR reactions were performed for selected genes (Table 1 ) according to the manufacturer’s instructions using 2 × MESAGREEN qPCR MasterMix Plus for the SYBR 258 Assay (Eurogentech, Liège, Belgium) in a 96-well qPCR plate (Sarstedt, Nümbrecht, Germany), and the Mx3005PTM sequence detection system (Agilent Technologies, Santa Clara, CA, USA). A quantitative analysis was made based on the cycle threshold (Ct) value for each well and calculated using MxPro software (Agilent Technologies, Santa Clara, CA, USA). The results were normalized using the mean Ct of the three housekeeping genes (Ct HKG) (Table 1 ) and the data are represented as dCt or as fold differences using the 2−ddCt method, where dCt = Ct target gene − Ct HKG.
Mx3005p sequence detection system
Manufactured by Agilent Technologies
Sourced in France, United States
The MX3005p sequence detection system is a real-time PCR instrument designed for gene expression analysis, genotyping, and other molecular biology applications. The system incorporates a high-performance optical detection module, thermal cycling capabilities, and intuitive software for data analysis and result interpretation. The MX3005p provides researchers with a reliable and efficient platform for their nucleic acid quantification needs.
Automatically generated - may contain errors
Lab products found in correlation
96 well qpcr plate, by Sarstedt (4 mentions)
Mxpro software, by Agilent Technologies (4 mentions)
Taqman pre designed gene expression assays, by Thermo Fisher Scientific (2 mentions)
Genorm version 3, by Primerdesign (2 mentions)
Nucleospin rna 2 extraction system, by Macherey-Nagel (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
9 protocols using mx3005p sequence detection system
1
Total RNA Extraction and RT-PCR Analysis
2
Quantitative Real-Time PCR Analysis
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The SuperscriptTM II Transcriptase Reverse Kit is used for RT (Invitrogen, Carlsbad, CA, USA) GB. Reverse transcription was performed using 1 µg of total RNA. The RT-PCR reactions were performed, for selected genes (Table 1 ), according to the manufacturer’s instructions using 2× MESA GREEN qPCR MasterMix Plus for SYBR 258 Assay (Eurogentech, Liège, Belgique) 96-well qPCR plate (Sarstedt, Nümbrecht, Germany), optical seal (Dutcher, Brumath, France), and the Mx3005PTM sequence detection system (Agilent technologies, Santa Clara, CA, USA). In each reaction, 10 ng of reverse transcripted RNA (based on initial RNA concentration) was used. All primers were used at 400 nM in a 20 µL reaction. Quantitative analysis was made based on the cycle threshold (Ct) value for each well and calculated using the MxPro software (Agilent technologies, Santa Clara, CA, USA). The results are normalized by three housekeeping (HKG) genes: 18S, GAPDH, and HPRT (Table 1 ), and data are represented as fold differences by the 2−ΔΔCt method, where ΔCt = Ct target gene—Ct HKG.
3
Reverse Transcription and qPCR Analysis
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The SuperscriptTM II Transcriptase Reverse Kit was used for RT (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using 1 µg of total RNA. The RT-PCR reactions were performed, for selected genes (Table 2 ), according to the manufacturer’s instructions using a 2X MESA GREEN qPCR MasterMix Plus for SYBR 258 Assay (Eurogentech, Seraing, BELGIUM), a 96-well qPCR plate (Sarstedt, Nümbrecht, Germany), an optical seal (Dutcher, Brumath, France), and the Mx3005PTM sequence detection system (Agilent technologies, Santa Clara, CA, USA.). In each reaction, 10 ng of reverse transcripted RNA (based on initial RNA concentration) was used. All primers were used at 400 nM in a 20 µL reaction. Quantitative analysis was carried out based on the cycle threshold (Ct) value for each well and calculated using the MxPro software (Agilent technologies, Santa Clara, CA, USA). The results were normalized by three housekeeping (HKG) genes: 18S, GAPDH, and HPRT (Table 2 ) and data are represented as fold differences by the 2−ΔΔCt method [23 (link)], where ΔCt = Ct target gene – Ct HKG.
4
Quantitative RT-PCR Analysis of Hk Genes
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The RT-PCR reactions were performed, for selected Hk genes (Table 2), according to the manufacturer's instructions using 2X MESA GREEN qPCR MasterMix Plus for SYBR® 258 Assay (Eurogentech, Liege, Belgium), 96 well qPCR plate (Sarstedt, Hampton, NH, USA), optical seal (Dutcher, Brumath, France) and the Mx3005PTM sequence detection system (Agilent technologies, Santa Clara, CA, USA). In each reaction, 10ng of reverse transcripted RNA (based on initial RNA concentration) was used. All primers were used at 400nM in a 20µL reaction. Quantitative analysis was made based on the cycle threshold (Ct) value for each well and calculated using MxPro software (Agilent Technologies, Santa Clara, CA, USA).
5
Optimized Real-Time PCR Quantification
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Real-time PCR was performed using Pre-designed Taqman gene expression assays (Applied Biosystems, Foster City, CA) for all target and housekeeping genes on MX3005p sequence detection system (Agilent). The TaqMan assay IDs are in Table 1 . To determine the stability and optimal number of housekeeping genes, we used geNORM version 3.4 (PrimerDesign) according to the manufacturer’s instructions (Vandesompele et al., 2002 (link)) and tested 12 commonly used reference genes of different functional classes in 10 samples from each test group. The average gene-stability measure (M) ranked β-actin and GAPDH as the most stable genes in our samples. PCR efficiency was tested over 5-log dilution series and confirmed that β-actin, GAPDH, and SKA2 had similar amplification efficiencies. For each primer/probe set, the PCR reaction was carried out using 10 µL of cDNA diluted 1:10-fold. Each quantitative PCR plate included a “no reverse transcriptase” and “no template” control to eliminate nonspecific amplification, one sample was run on a gel to confirm specificity, and samples were run in triplicates. Target gene quantitative PCR data was normalized to the geometic mean of β-actin and GAPDH and was expressed relative to the control samples using 2−(ΔΔCt) method.
6
RNA Extraction and Real-Time PCR Analysis
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Total RNA was isolated with the NucleoSpin RNA II extraction system (Macherey-Nagel) and reverse transcribed into cDNA. Gene expression was quantified by real-time PCR using the Mx3005P Sequence Detection System (Agilent Technologies). Primers were designed with Primer3 software and are listed in Table S1 . Samples without enzyme in the reverse transcription reaction were used as negative controls. Unspecific signals caused by primer dimers were excluded by nontemplate controls and by dissociation curve analysis. β-Actin was used to normalize for the amounts of cDNA within each sample. Differences were calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification (Dees et al., 2020 (link)).
7
Quantitative Gene Expression Analysis
8
Quantifying Hepatic CYP3A mRNA in Rats
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To determine CYP3A mRNA content in rat liver, the liver samples were homogenized and the total RNA was extracted from liver samples using RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen). The concentration of total RNA was determined by UV spectra at 260/280 nm, and the purity was determined by calculating the ration of UV absorbance. 2 µg of total RNA in a total volume of 50 µL (1 µg/25 µL) was reverse transcribed into template cDNA using dNTP mix (1 mM), random primers (0.5 µg/mL), AMV RT (22 U/µL), MgCl2 (25 mM) and RT buffer (10×).
For qRT-PCR, forward and reverse primer sequences (Table 2 ) of CYP3A gene were used. In a Stratagene Mx3005P sequence detection system (Agilent Technologies Agilent, Santa Clara, CA, USA), 50 ng of cDNA/sample and 10 µM forward and reverse primers were added to a total reaction mixture of 20 µL of SYBR Select Master Mix (Invitrogen). GAPDH was applied as the house-keeping gene. All assays were performed in triplicate. After 2 min at 50 °C and 10 min at 95 °C, amplifications were achieved with 40 repeating cycles at 95 °C for 15 s and 60 °C for 1 min, followed by a dissociation stage at 95 °C for 1 min, 55 °C for 30 s and 95 °C for 30 s. The CYP3A mRNA levels of groups A−D were expressed as a ratio of induced to vehicle control group (group A).
For qRT-PCR, forward and reverse primer sequences (
9
Quantitative PCR Analysis of Depressed Brain
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