~30 brains of each genotype were used for RNA extraction. cDNA was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen Cat. No. 18080) and used as a template for q-PCR using primers for 5HT7 and RPL32 (reference gene). Primers for 5HT7 were designed with the following sequences: forward primer 5’-GCATGGTGCGGAAATTGAGG-3’; reverse primer 5’-ACGGATATGGCACACAGA-3’; and for RPL32: forward primer 5’-GGACAGTATCTGATGCCCAAC-3’; reverse primer 5 ‘-ATCTCGCCGCAGTAAAGGC-3 ‘. The mRNA levels of 5HT7 were measured using QuantiFast SYBR Green PCR kit (Qiagen Cat. No. 204054) on a Mastercycler realplex2 instrument (Eppendorf). The relative mRNA levels were calculated using the comparative delta CT method.
Mastercycler realplex2 instrument
Manufactured by Eppendorf
Sourced in United States, Germany
The Mastercycler realplex2 instrument is a real-time PCR system designed for quantitative gene expression analysis. It provides precise temperature control and data acquisition capabilities for reliable and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Trizol kit, by Merck Group (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Microdrop, by Thermo Fisher Scientific (1 mentions)
5 protocols using mastercycler realplex2 instrument
1
Quantifying 5HT7 mRNA Expression
2
Bone Compartment RNA Extraction and qPCR
3
Grapevine Gene Expression Profiling
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Expression levels of 16 genes were analyzed by qRT-PCR. This gene set included 12 defense-related genes and four genes involved in sugar transport or metabolism (Supplementary Material S2 ). Primers were designed using the Primer3 tool (Untergasser et al., 2012 (link)) and tested for their specificity and efficiency (>95% for all primers). Real-time quantitative PCR was carried out using the GoTaq qPCR MasterMix (Promega) and a Mastercycler realplex2 instrument (Eppendorf). In this work, four V. vinifera reference genes were tested, elongation factor EF1γ and EF1α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin, described in several previous reports (Reid et al., 2006 (link); Nicolas et al., 2013 (link); Camps et al., 2014 (link)). The stability of all these genes was tested in our biological conditions. GAPDH was finally selected as the best reference gene. Gene expression results were normalized to GAPDH as an internal standard (Supplementary Material S2 ). Results were expressed as relative gene expression using the 2–ΔCt method (Schmittgen and Livak, 2008 (link); Cardot, 2017 ). For all qRT-PCR analyses, the number of independent biological replicates is specified in the legend caption. For each independent experiment, three technical replicates have been performed.
4
RNA Extraction and RT-qPCR Analysis in Arabidopsis
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Total RNA was extracted from the frozen ground A. thaliana tissues according to Kay et al. (1987 (link)). RNA quantity and quality were verified by using a Microdrop (Thermo Fischer, Waltham, MA, USA) and an agarose gel. Total RNA was treated with DNAse I (Sigma-Aldrich, St. Louis, MO, USA), and the reverse transcription was performed by using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time quantitative PCR (RT-qPCR) was carried out by using the GoTaq qPCR Master Mix (Promega, Madison, WI, USA) with a Mastercycler realplex2 instrument (Eppendorf, Hamburg, Germany). The results of the target gene expression were normalized with the average Ct value of the reference gene, AtPP2a (At1g13320) (Czechowski et al., 2005 (link)). The results were expressed as the relative gene expression according to the 2−ΔCt method. Primers were designed by using Oligo 7 and tested for specificity and efficiency (≥90%). The primer sequences used in this study are listed in Supplementary Table 2 .
5
Bone Tissue Fractionation and Gene Expression Analysis
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Tibiae were dissected and soft tissues removed. Next, we cut off the distal and proximal ends of the tibiae. These correspond to subcortical trabecular rich regions and the growth plates. Following this, we centrifuged the bone and collected the bone marrow in a new Eppendorf tube. After completely removing the bone marrow, the remaining cortical bone was selected as being osteocyte‐rich. Total RNA was extracted from the trabecular‐rich and cortical fractions using a TRIzol kit (Sigma, Boldon, UK) followed by cDNA synthesis (from 1 μg of total RNA) using TaqMan® reverse transcription reagents (Life Technologies, Inc., Carlsbad, CA, USA). We used SYBR® Green Master Mix for reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR) and a Mastercycler® realplex2 instrument (Eppendorf, Hamburg, Germany). We calculated mRNA expression using the standard fold change formula 2−(ΔΔCt). The levels of mRNA expression were normalized to geometrical means with Gapdh and Hprt expression (Table 1 ) as internal controls (2−(ΔCt)) and then expressed as fold values compared with the vehicle‐treated mice (2−(ΔΔCt)). The RT‐qPCR primers are listed in Table 1 .
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