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.
Taqman probe hs00193435 m1
Manufactured by Thermo Fisher Scientific
The TaqMan probe HS00193435 m1 is a pre-designed, gene-specific probe that can be used for quantitative real-time PCR (qRT-PCR) applications. The probe is labeled with a fluorescent reporter dye and a quencher dye, enabling the detection and quantification of a specific target sequence.
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2 protocols using taqman probe hs00193435 m1
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Quantitative Analysis of HAS2 Knockdown
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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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