Fast tissue rna purification kit

Total RNA was extracted from human or murine placental tissues or cells by using Fast Tissue RNA Purification Kit (EZBioscience, EZB-RN5), which was then reverse-transcribed into cDNA with 4×Reverse Transcription Master Mix (EZBioscience, A0010GQ). Quantitative reverse transcription-PCR (RT-qPCR) was performed using 2× Color SYBR Green qPCR Master Mix (ROX2 plus) (EZBioscience, A0012-R2) and the analysis was based on ABI Prism 7900 Fast Sequence Detection system (Thermo Fisher Scientific). The fold change at the transcriptional level of the target genes was displayed by calculating the 2^-ΔΔCt method and each sample was run in triplicates. Relative mRNA expression levels were normalized to β-actin (ACTB). Sequences of each pair of primers was displayed in Table 1.
Moreover, EZ-press Tissue Direct PCR Kit (EZBioscience, EZB-TDP1) was used to genotype Il-27ra allele of dissected murine embryos as manufacturer's instructions. PCR products were then amplified with the following primers: Wild-type Forward: 5′-CGCAGAAAGTTCTCATCT-3′; Wild-type Reverse: 5′- TCATACAGTACCCATCCC-3′; Knockout Forward: 5′- ACCGTAAAGCACGAGGAA-3′. Knockout Reverse: 5′- GACCACCAAGCGAAACAT -3′. Next, 2% agarose gel in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA) was prepared for electrophoresis at 90 V for 60 min.

Medium was aspirated, and cells were washed twice with phosphate-buffered saline (PBS) at room temperature. RNA was extracted using a Fast Tissue RNA Purification Kit (EZBioscience, Roseville, MN, USA), in accordance with the instructions of the kit. The concentration and quality of the RNA were detected by IMPLEN N60 Touch (Implen, Munich, Germany). Next, cDNA was prepared using the RNA as a template and a PrimeScript™ RT Reagent Kit (Takara, Shiga, Japan), in accordance with the instructions of the manufacturer, and diluted 1:5 in double-distilled water before quantitative real-time PCR (qPCR) plate preparation. All qPCR reactions were performed in 96-well plates, using SYBR^(R) Premix Ex Taq^TM. ACTB (encoding β-actin) was used as the housekeeping gene in qPCR for HMC3 cells. Relative expression was calculated using the 2^−∆∆Ct method [29 (link)]. The primer sequences used are as follows (Table 1).