Mircute mirna qpcr detection kit sybr

Peripheral arterial blood from patients with sepsis was collected within 24 h after their diagnosis. All samples were anticoagulated with ethylenediaminetetraacetic acid. Whole blood was treated with erythrocyte lysis buffer. For each 200-μL sample of blood, 1 ml TRIZOL (Tiangen Biotech Company, Beijing, China) was added and stored at − 80 °C. Blood cells from fresh peripheral blood were sorted into monocytes, lymphocytes, and neutrophils using flow cytometry (FCM). Total RNA was extracted from 200 μL of the blood sample, which had been stored at − 80 °C, as described above. miRNA levels in the total RNA were detected by real-time quantitative PCR (qRT-PCR) using a miRcute miRNA first-strand cDNA synthesis kit and miRcute miRNA qPCR detection kit (SYBR) (Tiangen Biotech Company). RNA levels were detected using a FastQuant cDNA synthesis kit and a SuperReal qRT-PCR detection kit (Tiangen Biotech Company). All procedures followed the manufacturer’s instructions. qRT-PCR was performed in triplicate. To normalize the expression levels of miRNAs, we used 5S rRNA as an internal control. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.

MiRNA was extracted from serum and tissue samples as described previously45 (link) with some modifications. The One Step Prime Script miRNA cDNA (Perfect Real-Time) Kit (TAKARA Biotechnology Co., Ltd, Dalian, China) was used to generate cDNA and the miRcute miRNA qPCR detection kit (SYBR) (TIANGEN Biotech Co., Ltd, Beijing, China) was used for real-time PCR analysis with the primer 5′-CGCGTTCACCGTGGCTAAGTTCC-3′. The methods used have been described previously46 (link)47 (link).

The miRNA extracted method from the serum and tissue was used as described by the literature [29] (link) with some modifications. The reverse transcription reaction was used One Step Prime Script miRNA cDNA (Perfect Real Time) Kit (TAKARA Biotechnology Co., Ltd, Dalian, China) and Real-time PCR reaction was used miRcute miRNA qPCR detection kit (SYBR) (TIANGEN Biotech Co., Ltd, Beijing, China). The detailed materials were shown in the Supplementary Methods.