Sybr premix ex taqtm 2 real time pcr kit

Total RNA was extracted with Trizol reagent (Invitrogen, California, USA) according to the manufacturer’s instructions. Then, cDNA was generated using a Reverse Transcriptase kit (Takara, Kusatsu, Japan). Then, the cDNA was used as template to determine the level of mRNA expression. The specific primers for Gli1 and GAPDH were as follows: Gli1 5ʹ-GGAAGTCATACTCACGCCTCGA-3ʹ (Forward), 5ʹ-CATTGCTGAAGGCTTTACTGCA-3ʹ (Reverse); GAPDH 5ʹ-AGAAGGCTGGGGCTCATTTG-3ʹ (Forward), 5ʹ-AGGGGCCATCCACAGTCTTC-3ʹ (Reverse). The quantitative real-time PCR (qRT-PCR) reactions were carried out in a Mx3000P System (Agilent Technologies, CA, USA) using a SYBR Premix Ex TaqTM II real-time PCR kit (Takara, Kusatsu, Japan). Each reaction was assessed in triplicate, and GAPDH was regarded as the internal control to normalize the results.

Total RNA was extracted from cells using the Trizol Reagent Kit (Invitrogen, California, USA). Then the total RNA was reverse transcribed into complementary DNA (cDNA) using Reverse Transcriptase kit (Takara, Kusatsu, Japan). Followed, the cDNA was used as template to determine the level of mRNA expression. The sequences of the primers were as follows: FBI-1 5ʹ-TTGCCAAAGATACCTGCTGA-3ʹ (Forward), 5ʹ-AAACCCCAAACAACCAAACA-3ʹ (Reverse); GAPDH 5ʹ-AGAAGGCTGGGGCTCATTTG-3ʹ (Forward), 5ʹ-AGGGGCCATCCACAGTCTTC-3ʹ (Reverse). The quantitative real-time PCR reactions were performed on the Mx3000P System (Agilent Technologies, CA, USA) using SYBR Premix Ex TaqTM II real-time PCR kit (Takara, Kusatsu, Japan). All samples were amplified in triplicate in one assay run simultaneously. GAPDH was regarded as the internal control to normalize the results.