Taqman r gene expression assay

Tissue samples were obtained either before or after trastuzumab-based therapy from patients with HER2-positive breast or gastric cancer who were previously treated by the Kindai University Faculty of Medicine after the study was approved by the Institutional Review Board; the patients provided written informed consent. Total RNA was isolated from paraffin-embedded tissues using the RNeasy Mini Kit and the RNeasy FFPE kit (both from Qiagen, CA, USA) according to the manufacturer's instructions. cDNA was prepared using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, CA, USA), and real-time PCR was conducted to assess the expression of heregulin, along with β-actin as an internal standard, using TaqMan (R) Gene Expression Assays (Applied Biosystems, CA, USA). Primers for heregulin (Hs00247620_m1) and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific (MA, USA). Fluorescence was detected using a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA). The final results were calculated using the ΔΔCt method, normalized to the levels of β-actin as an internal control, and standardized to the median value for each sample. PCR efficiency was evaluated via serial dilution of SK-BR-3 HRG cell cDNA, and the efficiencies were 93% and 91% for β-actin and heregulin, respectively.

RNA was extracted from NSps using TRIzol and pipet homogenization. Glycogen was added to the TRIzol sample after homogenization to aid in RNA recovery. RNA quantification was assessed using Epoch Biotek spectrophotometer (Biotek Instruments, Winooski, VT, USA). Utilizing the RT2 First Strand Kit (QIAGEN, Germantown, MD, USA), a 0.5 µg RNA sample was reverse-transcribed into cDNA. A 25 ng cDNA sample was then used as a template for RT-qPCR, employing TaqManR gene expression assays (Applied Biosystems, Foster City, CA, USA) (Supplementary Table S1) in the Applied Biosystems StepOnePlus Real-Time PCR System. Samples were run in duplicate for target genes and were normalized using HPRT1 as an endogenous control. Relative quantification of transcript expression was performed using the 2^−ΔΔCt method, where Ct represents the threshold cycle.

RNA was isolated using the RNeasy Micro Kit (Qiagen, Valencia, California, USA) and reverse transcribed. Each target cDNA was amplified via PCR on the same plate by using the TaqMan(R) Gene Expression Assays (Thermo Fisher Scientific) and the ABI 7300 Real-Time PCR System (Thermo Fisher Scientific). The Taqman probe used was derived from HIF-1α (Thermo Fisher Scientific, Assay ID; Hs00936376_ml, amplicon length 77). The relative amounts of target genes were determined in reference to 18S rRNA. Comparative threshold cycle (C_T) analysis was used to quantify transcripts. The value was calculated by the expression 2^−ΔΔCT.