One step tb green primescript rt pcr kit perfect real time

We totally collected 60 BC samples from patients and 30 normal breast tissues who underwent surgical treatments in Shengjing Hospital of China Medical University from 2007 to 2021. In these 60 BC samples, they were HR-, HER2+++ on immunohistochemistry or HR-, HER2++ on immunohistochemistry with fluorescence in situ hybridization (FISH) indicating that HER2 was amplified. Formalin fixation and paraffin embedding (FFPE) were to preserve the specimens. The study was approved by the hospital institutional ethics review committee. For evaluating the expression levels of 12 miRNA, we deparaffinized these specimens using xylene and ethanol. According to the manufacturer’s protocol, we extracted total RNA (including miRNAs) from FFPE tissue samples using TRIzol (Thermo Fisher Scientific, US), and cDNA synthesis was carrying out by using Mir-X miRNA qRT-PCR SYBR Kits (Takara Bio Inc., Kusatsu, Japan). Then, we performed real-time PCR reaction using One Step TB Green^® PrimeScript™ RT-PCR Kit (Perfect Real Time) (Takara Bio Inc., Kusatsu, Japan) on The LightCycler 480 Real-Time PCR System. 12 miRNAs expression levels were calculated by the 2^-ΔΔCt method and the cycle threshold (CT) values of miRNAs were normalized to the level of U6 as internal reference. Primers sequences used in our study were shown in table (Supplementary Table S2).

From the cluster a, cluster b, and cluster c networks, the top 9, 10, and 5 target genes were selected according to degree score values, and 15 miRNAs were randomly screened from the transcriptome sequencing results. Primers for genes and miRNAs were designed by NCBI Primer-Blast [32 (link)] and miRprimer software [33 (link)], respectively. All primer sequences were provided in S10 Table. mRNA and miRNA quantification was performed according to the instructions of the One Step TB Green^® PrimeScript^™ RT-PCR Kit (Perfect Real Time) (TaKaRa) and Mir-X^™ miRNA qRT-PCR TB Green^™ Kit (Clontech), respectively. PCR was performed on a LightCycler 96 (Roche) instrument. The reaction conditions were as follows: denaturation, 95 °C for 10 s; amplification, 40 cycles of 95 °C for 5 s and 60 °C for 20 s; and melting curve construction, 95 °C for 60 s, 55 °C for 30 s and 95 °C for 30 s. All experiments were repeated independently 3 times, and the results were calculated using the 2^-ΔΔCt method [34 (link)]. Tukey's honestly significant difference (HSD) test was used to test the significance of mRNA expression. The qRT-PCR results were visualized using GraphPad 7.0 (GraphPad Software, USA).