iTaq Universal SYBR Green PCR Kit (BioRad, Hercules, CA) was used for all assays and qPCR was performed in CFX Connect Real Time PCR system (Bio Rad, Hercules, CA). mRNA expression was normalized to β-Actin (ACTB) mRNA. The total reaction volume was 10 μL, including 10 μL 2X iTaq Universal SYBR Green mix, 10 μM left primer and 10 μM right primer, and 1 μL cDNA template. Each assay was performed in triplicate. Non template control (NTC) was added each time in all the assays. The PCR program started with 1 min. at 95°C, followed by 40 cycles (95°C, 30 s.; 57°C, 30 sec) and extension of 72°C for 2 min. The last steps of PCR are performed to acquire the dissociation curve and to validate the specificity of PCR amplicons.
Itaq universal sybr green pcr kit
Manufactured by Bio-Rad
The Bio-Rad ITaq Universal SYBR Green PCR Kit is a reagent system designed for real-time PCR amplification and detection. The kit contains all the necessary components, including the ITaq DNA polymerase, SYBR Green I dye, and buffer system, to perform quantitative PCR analysis.
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2 protocols using itaq universal sybr green pcr kit
1
Quantitative RT-PCR Assay for Gene Expression
2
Quantification of miRNA and mRNA Expression
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Trizol reagent (Invitrogen) was used for total RNA isolation. Quantification of miR-582-5p was performed as described previously [14 (link)]. cDNA was reverse-transcribed from RNA using the miRNA Reverse Transcription kit (Vazyme Biotech, Nanjing, China). The primers were as follows: miR-582-5p, forward: 5′-GCACACATTGAAGAGGACAGAC-3′, reverse: 5′-TATTGAAGGGGGTTCTGGTG-3′. For analysis of mRNA expression, cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). PCR was performed using the iTaq Universal SYBR Green PCR Kit (BioRad). The primers were as follows: CMTM8, forward: 5′-GGAGGAGCCGCAGCGCG-3′, reverse: 5′-CTGTATGGTCCTGGATCTCC-3′; KLF15, forward: 5′-ATGCACAAATGTACTTTCCCT-3′, reverse: 5′-TCAGTTCACGGAGCGCACGGA-3′; FOXG1, forward: 5′-CTTCATCCTGAGTCCCTACCG-3′, reverse: 5′-GCCGTTCTGCTGCATTCG-3′. Relative gene expression was analyzed using the 2−ΔΔCT method [19 (link)].
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