To measure the level of mRNA expressions, real-time PCR was performed using the StepOnePlus system (Applied Biosystems, Foster City, CA) as described previously (Haraguchi et al., 2010 (link); Haraguchi et al., 2012b (link); Haraguchi et al., 2012b (link); Haraguchi et al., 2015 (link); Nozaki et al., 2018 (link)). The sequence of each primer is shown in Table 1 . Gapdh was used as the internal standard. The reaction mixture contained SYBR Green Real-Time PCR Mix (Toyobo, Osaka, Japan), 400 nM of forward and reverse primers, and 30 ng of cDNA in a final volume of 20 μL. PCR was run with a standard cycling program: 95°C for 3 min; 40 cycles of 95°C, 15 s; 60°C, 15 s; and 72°C, 15 s. An external standard curve was generated by a serial 10-fold dilution of cDNA obtained from the salmon brain, which had been purified, and its concentration was measured. To confirm the specificity of the amplification, the PCR products were subjected to melting curve analysis and gel electrophoresis. The results were normalized to the expression of gapdh using StepOnePlus 2.0 software (Applied Biosystems).
Stepone plus 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The StepOne Plus 2.1 software is a real-time PCR analysis software from Thermo Fisher Scientific. It is designed to support the StepOne Plus real-time PCR system, providing data analysis and visualization capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Abi copycaller 2, by Thermo Fisher Scientific (3 mentions)
Sybr green real time pcr mix, by Toyobo (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (1 mentions)
Taqman copy number assay, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
5 protocols using stepone plus 2
1
Real-Time PCR for mRNA Expression Analysis
2
Epigenomic Profiling of Purkinje Cells
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Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP plus sonication chromatin IP kit (Cell Signaling Technology) according to the manufacturer’s protocol. Briefly, the Purkinje cell layer, including Purkinje cells, was laser-microdissected (LS-AMD; Leica Microsystems, Bensheim, Germany). The crosslinked cells of the Purkinje cell layer were sonicated to shear the chromatin to 200–1,000 bp. Each IP was performed using rabbit polyclonal anti-trimethyl-histone H3 (Lys9) antibody (07–442; Merck Millipore, Schwalbach, Germany), rabbit polyclonal anti-trimethyl-histone H3 (Lys27) antibody (07–449; Merck Millipore), or rabbit polyclonal anti-trimethyl-histone H4 (Lys20) antibody (07–463; Merck Millipore). qPCR analyses were performed using the StepOnePlus 2.0 software (Applied Biosystems) as described above. The sequence of each primer is shown in Table 1 .
3
E2F1 Copy Number Variation Analysis
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Copy number variation was evaluated on 20 ng of genomic DNA. Quantitative real-time polymerase chain reaction (PCR) TaqMan Copy Number Assays were performed using three probes targeting different regions of the E2F1 gene (Hs00576444_cn, Hs01758822_cn and Hs00919582_cn)(Applied Biosystems, Foster City, CA, USA). TaqMan CNV reactions were performed in triplicate using the FAM-dye-labeled assay for E2F1 and VIC-dye labeled RNase P assay. Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used for data analysis. Two independent assays were performed for each sample to confirm results.
4
Quantifying E2F1 Copy Number Variation
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Genomic DNA of patients was provided by the above-mentioned biobank. DNA was isolated from peripheral blood leucocytes using QIAamp DNA Blood Mini Kit, according to the manufacturer’s protocol (Qiagen Inc., Hilden, Germany). Copy number variation was evaluated on 20 ng of genomic DNA. Quantitative real-time polymerase chain reaction (PCR) TaqMan Copy Number Assays were performed using three probes targeting different regions of the E2F1 gene (Hs00576444_cn, Hs01758822_cn and Hs00919582_cn)(Applied Biosystems, Foster City, CA, USA). TaqMan CNV reactions were performed in triplicate using the FAM-dye-labeled assay for E2F1 and VIC-dye labeled RNase P assay. RNase P assay was used to normalize the genomic DNA input.
An internal DNA resulted with two copies of E2F1 both by TaqMan Copy Number Assay and array CGH was used as calibrator. Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used for data analysis. Copy Number ranging from 1.5 to 2.5 were predicted as CNV = 2. Two independent assays were performed for each sample to confirm results.
An internal DNA resulted with two copies of E2F1 both by TaqMan Copy Number Assay and array CGH was used as calibrator. Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc, Waltham, MA, USA) was used for data analysis. Copy Number ranging from 1.5 to 2.5 were predicted as CNV = 2. Two independent assays were performed for each sample to confirm results.
5
E2F1 Copy Number Quantification
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Quantitative real-time polymerase chain reaction (PCR) was performed using the TaqMan Copy Number Assay for E2F1 (Hs00576444_cn) (Applied Biosystems).
TaqMan CNV reactions were performed in triplicate using the FAM-dye-labeled assay for E2F1 and VIC-dyelabeled RNase P assay as a reference gene as described elsewhere (Jorgez et al. 2015) (link). Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc) was used for data analysis. Samples were run at least twice in independent assays to confirm results.
TaqMan CNV reactions were performed in triplicate using the FAM-dye-labeled assay for E2F1 and VIC-dyelabeled RNase P assay as a reference gene as described elsewhere (Jorgez et al. 2015) (link). Real-time data were collected by the StepOne Plus 2.1 software, and ABI CopyCaller 2.0 software (Thermo Fisher Scientific Inc) was used for data analysis. Samples were run at least twice in independent assays to confirm results.
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