After 48 h from siRNA transfection, total cellular RNA was isolated from SUM-149, MDA-MB 231, and MDA-MB-468 cells using innuPREP RNA mini-Kit (Analytik Jena AG, cat. no. 845-KS-2040050, Jena, Germany) and reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat. no. 4368814, Foster City, CA, USA). Quantitative real-time PCR was performed in duplicate for each target gene using RT2 SYBR Green qPCR Primer Assay (Qiagen, cat. no. 330500, Hilden, Germany) and Takyon™ ROX probe qPCR Kit (Eurogentec GmbH, cat. no. UF-RPMT-B0100, Cologne, Germany) in a 7300 real-time PCR detection system (Applied Biosystems). The relative gene expression levels were assessed using the 2−ΔΔCt method after normalization to the β-actin gene as an internal control. Melting curve analysis was conducted to confirm specific product amplification. The following target genes were assessed: VEGF-A, F3, F7, IGFBP1, IGFBP2, EDN1, F2R, and F2RL1. Primer sequences are listed in Supplementary Table S1 .
Rt2 sybr green qpcr primer assay
Manufactured by Qiagen
The RT2 SYBR Green qPCR Primer Assay is a pre-designed and optimized primer set for quantitative real-time PCR (qPCR) analysis. It is designed to detect and quantify target gene expression using the SYBR Green detection method.
Automatically generated - may contain errors
Lab products found in correlation
7300 real time pcr detection system, by Thermo Fisher Scientific (4 mentions)
Innuprep rna mini kit, by Analytik Jena (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Taqman probe hs00193435 m1, by Thermo Fisher Scientific (2 mentions)
Takyon rox probe qpcr kit, by Eurogentec (1 mentions)
4 protocols using rt2 sybr green qpcr primer assay
1
Quantitative Real-Time PCR Analysis of Gene Expression in Breast Cancer Cell Lines
2
Quantitative Gene Expression Analysis of Syndecans
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Quantitative real-time PCR (qPCR) for both cell lines was performed, RT2 SYBR Green qPCR Primer Assay (Qiagen, cat. no. 330500, Hilden, Germany) and Takyon™ ROX probe qPCR Kit (Eurogentec GmbH, cat. no. UF-RPMT-B0100, Cologne, Germany) in a 7300 real-time PCR detection system (Applied Biosystems, Darmstadt, Germany). The relative gene expression levels were assessed using the 2−ΔΔCt method after normalization to GAPDH gene expression as an internal control. Melting curve analysis was conducted to confirm specific product amplification. Gene expression of SDC1, SDC2, and SDC4 was analyzed using the TaqMan probes Hs00174579 m1, Hs00161617 m1, and Hs00299807 m1 (Applied Biosystems, Darmstadt, Germany), respectively. mRNAs analyzed and primer sequences (confirmed by NCBI BLAST analysis) are listed in Table 2 .
3
Quantitative Analysis of HAS2 Knockdown
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Total RNA was isolated from cells using the InnuPREP RNA mini kit (Analytikjena, cat. no. 845-KS-2040250, Jena, Germany). It was transcripted into cDNA carried out with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat. no. 4368814, Foster City, CA, USA) following the supplier´s protocols. qPCR was performed in a 7300 real-time PCR detection system (Applied Biosystems) with RT2 SYBR Green qPCR Primer Assay (Qiagen, cat. no. 330500, Hilden, Germany) and Takyon™ ROX probe qPCR Kit (Eurogentec GmbH, cat. no. UF-RPMT-B0100, Cologne, Germany). HAS2 knockdown was confirmed using the TaqMan probe HS00193435 m1 (Applied Biosystems). Results were evaluated using the 2−∆∆Ct method. β-actin samples were used as internal controls. The fold change shows the expression of the investigated enzymes in HAS2 knockdown cells compared to the control samples. Primer sequences are shown in Supplementary Table S1. The results were formed out of 7 experiments with double or triple replicates in each experiment.
4
Quantitative Analysis of HAS2 Knockdown
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The RNA of SKOV3 cells was isolated using the InnuPREP RNA mini kit (Analytikjena, cat. no. 845-KS-2040250, Jena, Germany) . It was transcripted into cDNA carried out with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat. no. 4368814, Foster City, CA, USA) following the supplier´s protocols. Quantitative real-time PCR (qPCR) was performed in a 7300 real-time PCR detection system (Applied Biosystems) with RT2 SYBR Green qPCR Primer Assay (Qiagen, cat. no. 330500, Hilden, Germany) and Takyon™ ROX probe qPCR Kit (Eurogentec GmbH, cat. no. UF-RPMT-B0100, Cologne, Germany). HAS2 knockdown was confirmed using the TaqMan probe HS00193435 m1 (Applied Biosystems).
Results were evaluated using the 2 -∆∆Ct method. Beta-actin samples were used as internal controls. The fold change shows the expression of the investigated enzymes in HAS2 knockdown cells compared to the control samples. Primer Sequences are shown in Supplementary table I. The results were formed out of 7 experiments with double or triple replicates in each experiment.
Results were evaluated using the 2 -∆∆Ct method. Beta-actin samples were used as internal controls. The fold change shows the expression of the investigated enzymes in HAS2 knockdown cells compared to the control samples. Primer Sequences are shown in Supplementary table I. The results were formed out of 7 experiments with double or triple replicates in each experiment.
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