Quantitative real-time PCR was performed as described previously (Elbadawy et al., 2020a (link)). Briefly, RNA samples were prepared from Mdivi-1- or DMSO-treated NLO and minced fragments from precooled liver tissue in liquid nitrogen using the NucleoSpin RNA kit (MACHEREY-NAGEL, Düren, Germany). The RNA was then converted to cDNA using the ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan). The PCR was then performed on cDNA using the QuantiTect SYBR I kit (Qiagen, Hilden, Netherlands) and the StepOnePlus Real-Time PCR system (Applied Biosystems, Waltham, MA, United States). Using the 2−ΔΔCT method, values of cycle threshold (Ct) obtained in quantification were used for calculations of fold changes in mRNA abundance. The specific primers (Fasmac Corporation, Kanagawa, Japan) used for experiments were shown in Table 1 .
Quantitect sybr 1 kit
Manufactured by Qiagen
Sourced in Netherlands
The QuantiTect SYBR I kit is a real-time PCR reagent designed for the detection and quantification of DNA sequences. The kit contains a SYBR Green-based master mix that enables the amplification and fluorescent detection of target DNA during the PCR process. The core function of the kit is to provide a versatile and reliable solution for quantitative gene expression analysis in a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Nucleospin kit, by Takara Bio (2 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Quantitect reverse transcription kit, by Toyobo (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Nucleospin rna kit, by Takara Bio (1 mentions)
4 protocols using quantitect sybr 1 kit
1
Quantitative Real-Time PCR for mRNA Quantification
2
Quantitative Real-Time PCR Analysis of Canine BC Stem Cell Markers
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Quantitative real-time PCR was performed as described previously14 (link). Total RNA was extracted from 2 × 105 of 2.5D organoid cells at early and late passage, 3D organoids, and urothelial carcinoma cell lines by using a NucleoSpin kit (Takara Bio Inc., Shiga, Japan) following the manufacturer’s protocol. First-strand cDNA was prepared using a QuantiTect Reverse Transcription Kit (TOYOBO, Tokyo, Japan). Quantitative real-time PCR was done using a QuantiTect SYBR I Kit (QIAGEN, Hilden, Germany) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The ΔΔCq method was used for quantification. Specific primers used for dog BC stem cell markers, SOX2, CD44, and GAPDH were designed and are shown in Table 3 .
Primers for real-time quantitative PCR analysis.
| Gene name | Primer | Sequence |
|---|---|---|
| SOX2 | Forward | 5′-GCCCTGCAGTACAACTCCAT-3′ |
| Reverse | 5′-GGAGTGGGAGGAGGAGGTAA-3′ | |
| CD44 | Forward | 5′-CCAAGACAGTTCCAGGGTGT-3′ |
| Reverse | 5′-TTGAGGTTTCCGCATAGGAC-3′ | |
| GAPDH | Forward | 5′-AACTCCCTCAAGATTGTCAGCAA-3′ |
| Reverse | 5′-CATGGATGACTTTGGCTAGAGGA-3′ |
3
Transcriptomic Analysis of Bladder Tissues
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Total RNA was extracted from normal bladder tissues, organoid samples, 2D urothelial carcinoma cell lines, and blood samples using the NucleoSpin kit (Takara Bio Inc) according to the manufacturer's instructions. First‐strand cDNA was synthesized using a QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative real‐time PCR was performed using a QuantiTect SYBR I Kit (QIAGEN) and a StepOnePlus Real‐Time PCR System (Applied Biosystems). The ΔΔCq method was used for quantification. Specific primers used for dog CK5, DSG3, GATA3, ERBB2, MMP28, CTSE, CNN3, TFPI2, COL17A1, AGPAT4, GAPDH, CK15, TGM2, GJB2, IL1R2, COL5A2, CDH3, CHST4, and ADORA2B are listed in Table 2 .
4
Chaga's Impact on Cell Cycle and CSC Pathways
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The effects of Chaga on cell cycle- and CSC-related signal pathways in DBCO were assayed using quantitative real-time PCR. Concisely, the organoids were cultured on a 24-well culture plate at 1 × 105 cells/well. Twenty-4 h later, the organoid cells were treated with Chaga (100 μg/ml), or DMSO (for the control wells) for 24 h. Thereafter, organoids were then harvested, and the total RNA was generated using the NucleoSpin RNA kit (TAKARA, Tokyo, Japan) and converted to first-strand cDNA using the ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan) following the manufacturer’s protocols. Real-time PCR was carried out by the QuantiTect SYBR I kit (Qiagen, Hilden, Netherlands) and StepOnePlus Real-Time PCR system (Applied Biosystems, Waltham, Massachusetts, United States). The ΔΔCq method was used to quantify the data. Data analysis was carried out in triplicates and GAPDH was used as a control gene. The primers for GAPDH, Cyclin A2, Cyclin D1, Cyclin E1, CDK4, CD44, c-MYC, SOX2, and YAP1 genes were used (Fasmac, Table 2 ).
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