The cochlear tissue was dissected in cold-PBS and homogenized with the Qiazol lysis reagent (Qiagen, Hilden, Germany). A high pure RNA isolation kit (Roche Diagnostic, GmbH, Germany) was used to isolate the total RNA in the homogenate according to the manufacturer’s protocol. The RNA concentration was evaluated with a spectrophotometer (Shimadzu UV-mini 1240, GmbH, Germany). The complementary DNA (cDNA) was synthesized using a transcriptor high fidelity cDNA synthesis kit (Roche Applied Science, Penzberg, Germany). Each prepared sample used 20 μL of the SYBR green qPCR reaction kit (Roche Applied Science), which contained 2 μL of cDNA for qRT‐PCR using the following primer pairs: CASP-3, forward 5’-GGAGCAGCTTTGTGTGTGTG-3’ and reverse 5’-CTTTCCAGTCAGACTCCGGC-3’ ; BAX, forward 5’-GTTTCATCCAGGATCGAGCAG-3’ and reverse 5’-CATCTTCTTCCAGATGGTGA-3’ ; BCL-2, forward 5’-CCTGTGGATGACTGAGTACC-3’ and reverse 5’-GAGACAGCCAGGAGAAATCA-3’ ; GAPDH, forward 5’-CTTCCGTGTTCCTACCCCCAATGT-3’ and reverse 5’-GCCTGCTTCACCACCTTCTTGATG-3’. The GAPDH sequences were used for normalization, as it represents a housekeeping gene. The rotor-gene Q 5plex HRM platform (Qiagen, Hilden, Germany) was used for the qRT-PCR, and the data were analyzed using the comparative Ct method (ΔΔCT).
Sybr green qpcr reaction kit
Manufactured by Roche
Sourced in Germany
The SYBR green qPCR reaction kit is a laboratory reagent designed for quantitative real-time polymerase chain reaction (qPCR) analysis. It contains a fluorescent dye, SYBR Green, which binds to double-stranded DNA, enabling the detection and measurement of DNA amplification during the PCR process.
Automatically generated - may contain errors
3 protocols using sybr green qpcr reaction kit
1
Quantitative RT-PCR Analysis of Cochlear Apoptosis
2
Quantitative Analysis of SETDB1 Expression
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Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the level of SETDB1 gene expression in the cell lines. A High Pure RNA Isolation Kit (Roche Diagnostics, USA) was used to isolate the RNA. For the qRT-PCR, a Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science, Germany) was used to synthesize complementary DNA (cDNA) in a thermal cycler. Briefly, 2 µL of cDNA was mixed with 18 µL from the SYBR Green qPCR reaction kit (Roche Applied Science, Germany) for the qRT‐PCR using primer pairs (Table 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used for normalization of the SETDB1 expression in qRT-PCR using the comparative CT method (ΔΔCT) (Livak and Schmittgen, 2001). qRT-PCR was carried as described in the manufacturer’s protocol (Rotor-Gene Q 5plex HRM Platform; QIAGEN, Germany) (Sun et al., 2014).
3
Quantitative Gene Expression Analysis of Cochlear Tissue
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The cochlear tissue was dissected in cold-PBS and homogenized with the Qiazol lysis reagent (Qiagen, Hilden, Germany). A high pure RNA isolation kit (Roche Diagnostic, GmbH, Germany) was used to isolate the total RNA in the homogenate according to the manufacturer's protocol. The RNA concentration was evaluated with a spectrophotometer (Shimadzu UV-mini 1240, GmbH, Germany). The complementary DNA (cDNA) was synthesized using a transcriptor high fidelity cDNA synthesis kit (Roche Applied Science, Penzberg, Germany). Each prepared sample used 20 µL of the SYBR green qPCR reaction kit (Roche Applied Science), which contained 2 µL of cDNA for qRT-PCR using the following primer pairs: CASP-3, forward 5'-GGAGCAGCTTTGTGTGTGTG and reverse 5'-CTTTCCAGTCAGACTCCGGC-3'; BAX, forward 5'-GTTTCATCCAGGATCGAGCAG-3' and reverse 5'-CATCTTCTTCCAGATGGT GA-3'; BCL-2, forward 5'-CCTGTGGATGACTGAGTACC-3' and reverse 5'-GAGACAGCCAGGAGAAATCA-3'; GAPDH, forward 5'-CTTCCGTGTTCCTACCCCCAATGT-3' and reverse 5'-GCCTGCTTCACCACCTTCTTGATG-3'. The GAPDH sequences were used for normalization, as it represents a housekeeping gene. The rotor-gene Q 5plex HRM platform (Qiagen, Hilden, Germany) was used for the qRT-PCR, and the data were analyzed using the comparative Ct method ( ΔΔ C T ).
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