Total RNA was isolated from cells using the RNeasy total RNA isolation kit (Qiagen, Hilden, Germany). cDNA synthesis was performed with 1 µg of total RNA using the MiScript RT-PCR system (Qiagen). The SYBR green quantitative PCR (qPCR) assay kit (Roche, Basel, Switzerland) was used with 2 µl of diluted cDNA for each sample. The forward and reverse primers for qPCR of murine MUC1, human PD-L1, DICER, c-Jun, miR-200c, miR-34a, premir-200c, pre-mir-34a and GAPDH are listed in Supplementary Table S2 . Statistical significant was determined by the student’s t-test.
Miscript rt pcr system
Manufactured by Qiagen
The MiScript RT-PCR system is a laboratory equipment product designed for the quantitative analysis of microRNA (miRNA) expression. It enables the detection and quantification of miRNAs using real-time PCR technology.
Automatically generated - may contain errors
2 protocols using miscript rt pcr system
1
Quantitative RNA Expression Analysis
2
Assessing Plasma RNA in Low-Concentration Samples
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The concentration of total RNA extracted from plasma was generally close to, or below, the manufacturer's stated detection limit of the Nanodrop 2000 spectrophotometer and the RNA 6000 Nanochip of the Agilent Bioanalyzer. The concentration of small RNA (i.e., from 5–150 nt) was also generally at background levels as measured by the Small RNA kit of the Agilent Bioanalyzer. The low RNA concentration precluded the reliable assessment of RNA purity by A260/A280 and A260/A230 absorbance ratios, and it was also too low for assessment of RNA integrity by Agilent Bioanalyzer "RIN" number. To identify samples that may potentially be contaminated with nucleases and/or RT/PCR inhibitors we utilized the integrated quality controls in the miScript RT-PCR system (Qiagen), which includes the miRNA reverse transcription control (miRTC) and positive PCR control (PPC). The miRTC and PPC reactions were performed for all subjects and were within the manufacturer's specified range.
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