Qpcr detection kit sybr green

Total RNA was extracted from the TM cells with a Total RNA Extraction Kit (Tiangen, Beijing, China) and reverse transcribed into cDNA with a Synthesis Kit (Takara, Shiga, Japan). QRT-PCR was carried out by using a qPCR Detection Kit (SYBR Green; Tiangen). Gene expression was normalized against GAPDH expression. The 2^−ΔΔCt method was used for calculation. All primer pairs are displayed in Table 2.Table 2

Primers used in the qPCR experiments.

Gene	Sequence
ANGPTL7-F	5′-CGGCTGCGTGTAGAGATGGA-3′
ANGPTL7-R	5′-CCTTGGTGCTGAAGGCTGTGT-3′
SP1-F	5′-GAGGGCAGGGTGCCAATG-3′
SP1-R	5′-TTCTGTAAGTTGGGAGCGGC-3′
GAPDH-F	5′-AATCCCATCACCATCTTC-3′
GAPDH-R	5′-AGGCTGTTGTCATACTTC-3′

To validate the veracity and reliability of the transcriptome data, 8 DEGs were randomly selected for conducting real-time qPCR validation. The upstream and downstream primers of these genes (Table S1) were designed using the NCBI’s Primer-BLAST and synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China). Using β-actin as an internal reference gene, real-time qPCR was performed on a 7900HT system (Applied Biosystem, Foster City, CA, USA) using a qPCR Detection kit (SYBR Green, TIANGEN, Beijing, China). Data were processed using 7900HT Software (v.2.0.6) (Applied Biosystem) and the relative quantification of the gene expression differences was calculated with the formula 2^−ΔΔCt.