Cfx connect unit

Manufactured by Bio-Rad

Sourced in China

The CFX-Connect is a real-time PCR detection system manufactured by Bio-Rad. It is designed for quantitative gene expression analysis, genotyping, and other real-time PCR applications. The system includes a thermal cycler and optical detection module to perform fluorescence-based real-time PCR experiments.

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Lab products found in correlation

M mulv reverse transcriptase, by New England Biolabs (14 mentions) Trizol reagent, by Thermo Fisher Scientific (12 mentions) Sybr green qpcr master mix, by Selleck Chemicals (6 mentions) Nucleozol reagent, by Takara Bio (3 mentions) Forget me not qpcr master mix, by Biotium (1 mentions) Dntps, by GenScript (1 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions)

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CFX Connect (733 citations)

16 protocols using cfx connect unit

Quantifying Gene Expression in Ovarian Cancer

Cited 5 times

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Subconfluent parental human epithelial ovarian cancer lines OVCAR8 and SKOV3 and cisplatin-resistance human epithelial ovarian cancer cell lines OVCAR8CR and SKOV3CR were treated with various conditions for 48 h. Total RNA was isolated from treated cells by using the NucleoZOL reagent (Takara Bio USA Inc) and subjected to reverse transcription (RT) reactions with hexamer, M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA), and dNTPs (GenScript USA Inc. Piscataway, NJ, USA). RT products were used as qPCR templates. The qPCR primers for the genes of interest were designed by using the Primer3 Plus program (Table S1). TqPCR reactions were carried out by using SYBR Green-based Forget-Me-Not™ qPCR Master Mix (Biotium Inc., Hayward, CA, USA) on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA, USA) as described.⁴² (link)^,49 , 50 (link), 51 (link), 52 (link), 53 (link)
GAPDH was used as a reference gene. Relative expression was calculated by using the 2^−ΔΔCt method. All qPCR reactions were done in triplicate.

Huang L., Zhang J., Deng Y., Wang H., Zhao P., Zhao G., Zeng W., Wang Y., Chen C., Wagstaff W., Haydon R.C., Reid R.R., He T.C., Shen L., Luu H.H, & Zhao L. (2023). Niclosamide (NA) overcomes cisplatin resistance in human ovarian cancer. Genes & Diseases, 10(4), 1687-1701.

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Quantitative RT-PCR Liver RNA Analysis

Cited 3 times

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Total RNA was isolated from both cells and freshly-prepared liver tissues by using the TRIZOL Reagent (Invitrogen, China) as described [55 (link), 56 (link)]. Briefly, fresh mouse liver samples at different development stages (n=5, CD1 male, each time point) or from the NAFLD model were dissected out, minced, and ground in TRIZOL Reagent. Similarly, primary mouse hepatocytes were treated with different conditions and lysed in TRIZOL Reagent for total RNA isolation. Total RNA was subjected to reverse transcription with hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were used as qPCR templates. The gene-specific PCR primers were designed by using Primer3 Plus (Supplementary Table 1). TqPCR was carried out by using 2x SYBR Green qPCR Master Mix (Bimake, Shanghai, China) on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [57 (link), 58 (link)]. All TqPCR reactions were done in triplicate. Gapdh was used as the reference gene. Quantification of gene expression was carried out by using the 2-ΔΔCq method as described [59 (link)].

Peng Q., Chen B., Wang H., Zhu Y., Wu J., Luo Y., Zuo G., Luo J., Zhou L., Shi Q., Weng Y., Huang A., He T.C, & Fan J. (2019). Bone morphogenetic protein 4 (BMP4) alleviates hepatic steatosis by increasing hepatic lipid turnover and inhibiting the mTORC1 signaling axis in hepatocytes. Aging (Albany NY), 11(23), 11520-11540.

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Total RNA Isolation and qPCR Analysis

Cited 2 times

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The total RNA was isolated from the cultured cells using TRIZOL Reagent (Invitrogen, China) as described [31 (link), 32 (link)]. Briefly, Huh7 and HepG2 cells treated with various conditions were lysed in TRIZOL Reagent for total RNA isolation. The total RNA was subjected to reverse transcription with hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were used as PCR templates. Gene-specific PCR primers were designed by using Primer 3 program. All primer sequences are shown in Table S2. TqPCR was carried out by using 2× SYBR Green qPCR Master Mix (Bimake, Shanghai, China) on the CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [32 (link)]. All TqPCR reactions were done in triplicate. GAPDH was used as a reference gene. Quantification of gene expression was carried out by using the 2^−ΔΔCq method as described [31 (link)].

Zhong J., Tian L., Gou Y., Zhao P., Dong X., Guo M., Zhao G., Li A., Hao A., He T.C, & Fan J. (2023). BMP4 upregulates glycogen synthesis through the SMAD/SLC2A1 (GLUT1) signaling axis in hepatocellular carcinoma (HCC) cells. Cancer & Metabolism, 11, 9.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA from freshly‐prepared liver tissues was isolated by using the TRIZOL Reagent (Invitrogen) as described.⁴⁵, ⁴⁶ Total RNA was subjected to reverse transcription with hexamer and M‐MuLV reverse transcriptase (New England Biolabs). The cDNA products were further diluted and used as PCR templates. Gene‐specific qPCR primers were designed by using the Primer3 program (Table S1). TqPCR was carried out by using 2x SYBR Green qPCR Master Mix (Bimake) on a CFX‐Connect unit (Bio‐Rad Laboratories) as described.⁴⁷ All TqPCR reactions were done in triplicate. Gapdh was used as a reference gene. Quantification of gene expression was carried out by using the 2^−ΔΔCq method as described.⁴⁸

Gou Y., Weng Y., Chen Q., Wu J., Wang H., Zhong J., Bi Y., Cao D., Zhao P., Dong X., Guo M., Wagstaff W., Hendren‐Santiago B., Chen C., Youssef A., Haydon R.C., Luu H.H., Reid R.R., Shen L., He T, & Fan J. (2022). Carboxymethyl chitosan prolongs adenovirus‐mediated expression of IL‐10 and ameliorates hepatic fibrosis in a mouse model. Bioengineering & Translational Medicine, 7(3), e10306.

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Optimized qPCR protocol for gene expression

Cited 1 time

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Total RNA was isolated using TRIZOL Reagents (Invitrogen) and reverse transcribed using hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The resulting cDNA products were diluted 10- to 100-fold and used as PCR templates. TqPCR was carried out by using SYBR Green-based TqPCR analysis on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA). PCR primers were designed by using Primer3 program and were listed in Supplementary Table 1. Quantitative real-time PCR analysis was carried out by using our recently optimized TqPCR protocol [82 (link)]. Briefly, the PCR reactions were carried out by using a touchdown protocol: 95°C×3min for one cycle; 95°C×20 sec, 66°C×10 sec for 4 cycles, with 3°C decrease per cycle; followed by 95°C×10 sec, 55°C×15 sec, 70°C×1 sec for 40 cycles, followed by plate read. All reactions were done in triplicate. The TqPCR amplification was confirmed by performing the melting curve test and observing a single peak for each gene. Gapdh was used as a reference gene.

Hu X., Li L., Yu X., Zhang R., Yan S., Zeng Z., Shu Y., Zhao C., Wu X., Lei J., Li Y., Zhang W., Yang C., Wu K., Wu Y., An L., Huang S., Ji X., Gong C., Yuan C., Zhang L., Liu W., Huang B., Feng Y., Zhang B., Haydon R.C., Luu H.H., Reid R.R., Lee M.J., Wolf J.M., Yu Z, & He T.C. (2017). CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs). Oncotarget, 8(67), 111847-111865.

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RNA Extraction and qPCR Analysis of Gene Expression

Cited 3 times

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Total RNA was isolated by using the TRIZOL Reagent (Invitrogen, China), and subjected to reverse transcription using hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were used as PCR templates. Gene-specific PCR primers were designed by using Primer3 program (Table S1). TqPCR was carried out by using 2x SYBR Green qPCR Master Mix (Bimake, Shanghai, China) on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [52 ,53 (link)]. All TqPCR reactions were done in triplicate. Gapdh was used as a reference gene. Quantification of gene expression was carried out by using the 2^−ΔΔCq method as described [32 (link),51 (link),[54] (link), [55] (link), [56] ]. Clustering heatmap analysis of relative gene expression was carried out by using the pheatmap package in R (4.2.2).

Gou Y., Huang Y., Luo W., Li Y., Zhao P., Zhong J., Dong X., Guo M., Li A., Hao A., Zhao G., Wang Y., Zhu Y., Zhang H., Shi Y., Wagstaff W., Luu H.H., Shi L.L., Reid R.R., He T.C, & Fan J. (2023). Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering. Bioactive Materials, 34, 51-63.

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Comprehensive RNA Isolation and Analysis

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Total RNA from both cultured cells and freshly prepared liver tissues was isolated by using the TRIZOL Reagent (Invitrogen) by following the manufacturer's instructions. Briefly, the freshly prepared mouse liver tissue at the indicated development stages (n = 5 CD1 male mice for each time‐point) or the NAFLD model was dissected out, minced and ground in the TRIZOL Reagent. Alternatively, cultured cells were lysed in TRIZOL Reagent for total RNA isolation. Total RNA was subjected to reverse transcription with hexamer and M‐MuLV reverse transcriptase (New England Biolabs). The cDNA products were used as PCR templates. The H19 RT primer and gene‐specific PCR primers were designed by using Primer3 program (Table S3). TqPCR was carried out by using 2× SYBR Green qPCR Master Mix (Bimake) on a CFX‐Connect unit (Bio‐Rad Laboratories) as described.39, 40, 41 All TqPCR reactions were done in triplicate. Gapdh was used as a reference gene. Quantification of gene expression was performed using the 2^−ΔΔCq method.42

Wang H., Cao Y., Shu L., Zhu Y., Peng Q., Ran L., Wu J., Luo Y., Zuo G., Luo J., Zhou L., Shi Q., Weng Y., Huang A., He T, & Fan J. (2019). Long non‐coding RNA (lncRNA) H19 induces hepatic steatosis through activating MLXIPL and mTORC1 networks in hepatocytes. Journal of Cellular and Molecular Medicine, 24(2), 1399-1412.

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Total RNA Isolation and Quantitative PCR

Cited 6 times

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Total RNA was isolated by using the TRIZOL Reagent (Invitrogen, China), and subjected to reverse transcription using hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were used as PCR templates. Gene-specific PCR primers were designed by using Primer3 program (Table S1). TqPCR was carried out by using 2x SYBR Green qPCR Master Mix (Bimake, Shanghai, China) on the CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [[36] , [37] (link), [38] (link), [39] (link)]. All TqPCR reactions were done in triplicate. Gapdh was used as a reference gene. Quantification of gene expression was carried out by using the 2^−ΔΔCq method as described [38 (link),[40] (link), [41] (link), [42] (link)].

Zhong J., Wang H., Yang K., Wang H., Duan C., Ni N., An L., Luo Y., Zhao P., Gou Y., Sheng S., Shi D., Chen C., Wagstaff W., Hendren-Santiago B., Haydon R.C., Luu H.H., Reid R.R., Ho S.H., Ameer G.A., Shen L., He T.C, & Fan J. (2021). Reversibly immortalized keratinocytes (iKera) facilitate re-epithelization and skin wound healing: Potential applications in cell-based skin tissue engineering. Bioactive Materials, 9, 523-540.

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Quantifying Ovarian Cancer Gene Expression

Cited 2 times

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Subconfluent ovarian cancer cells were treated with varied concentrations of monesin for 48 h. Total RNA was isolated from the treated cells by using TRIZOL Reagents (Invitrogen) and subjected to reverse transcription reactions with hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). Such cDNA products were used as PCR templates. The qPCR primers were designed by using Primer3 program69 (link) and used to amplify the genes of interest (Supplemental Table 1). The TqPCR were carried out by using the SYBR Green-based qPCR analysis on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA), as described24 (link). The qPCR reactions were done in triplicate. GAPDH was used as a reference gene.

Deng Y., Zhang J., Wang Z., Yan Z., Qiao M., Ye J., Wei Q., Wang J., Wang X., Zhao L., Lu S., Tang S., Mohammed M.K., Liu H., Fan J., Zhang F., Zou Y., Liao J., Qi H., Haydon R.C., Luu H.H., He T.C, & Tang L. (2015). Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells. Scientific Reports, 5, 17523.

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Quantitative PCR Analysis of MBZ-Treated Cells

Cited 2 times

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Subconfluent cells were treated with varied concentrations of MBZ for 48 h and subjected to total RNA isolation samples using TRIZOL Reagents (Invitrogen). Total RNA was also isolated from the MBZ or DMSO-treated CAL27-derived xenograft tumor samples using TRIZOL Reagents. The isolated RNA was subjected to reverse transcription reactions with hexamer and M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA). The cDNA products were used as PCR templates. The qPCR primers were designed by using Primer3 program [57 (link)], and used to amplify the genes of interest (Supplementary Table 1). TqPCR was carried out by using SYBR Green-based qPCR analysis on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) as described [29 (link)]. The qPCR reactions were done in triplicate. GAPDH was used as a reference gene.

Zhang F., Li Y., Zhang H., Huang E., Gao L., Luo W., Wei Q., Fan J., Song D., Liao J., Zou Y., Liu F., Liu J., Huang J., Guo D., Ma C., Hu X., Li L., Qu X., Chen L., Yu X., Zhang Z., Wu T., Luu H.H., Haydon R.C., Song J., He T.C, & Ji P. (2017). Anthelmintic mebendazole enhances cisplatin's effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC). Oncotarget, 8(8), 12968-12982.

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