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Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR
Published on: June 27, 2017
Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR
Keiji Hirai1, Hiromichi Yoshizawa2, Toshimi Imai2
1Division of Nephrology, First Department of Integrated Medicine, Saitama Medical Center, Jichi Medical University.
Abstract:
MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.
Insights
Researchers developed a new method to measure microRNA (miRNA) expression in rat peritoneal membranes. This technique identified increased miRNA levels in a rat peritoneal fibrosis model, aiding disease research.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- MicroRNAs (miRNAs) are crucial post-transcriptional regulators of gene expression.
- Investigating miRNA expression profiles is vital across various tissues.
- Established methods for miRNA purification and detection in rat peritoneal membranes are lacking.
Purpose of the Study:
- To establish a reliable protocol for miRNA purification and quantification in rat peritoneal membranes.
- To analyze miRNA expression profiles in a rat model of peritoneal fibrosis.
Main Methods:
- Developed a four-step protocol involving sample purification, RNA extraction, cDNA synthesis, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR).
- Applied the protocol to rat peritoneal membrane samples from control and fibrosis model groups.
Main Results:
- Successfully purified and quantified miRNAs from rat peritoneal membrane samples.
- Identified significant upregulation of six specific miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) in the peritoneal fibrosis model compared to controls.
Conclusions:
- The developed qRT-PCR protocol provides an effective and reliable method for studying miRNA expression in rat peritoneal membranes.
- This method facilitates the investigation of miRNA roles in various peritoneal pathologies, including fibrosis.
