Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

Keiji Hirai; Hiromichi Yoshizawa; Toshimi Imai; Yusuke Igarashi; Ichiro Hirahara; Susumu Ookawara; Kenichi Ishibashi; Yoshiyuki Morishita

doi:10.3791/55505

Rilevamento dell'espressione di microRNA nella membrana peritoneale dei ratti usando il quant...

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Genetics

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JoVE Journal Genetics

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

Rilevamento dell'espressione di microRNA nella membrana peritoneale dei ratti usando il quantitativo PCR in tempo reale

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08:56 min

June 27, 2017

DOI: 10.3791/55505-v

Keiji Hirai¹, Hiromichi Yoshizawa², Toshimi Imai², Yusuke Igarashi², Ichiro Hirahara², Susumu Ookawara², Kenichi Ishibashi³, Yoshiyuki Morishita¹

¹Division of Nephrology, First Department of Integrated Medicine,Saitama Medical Center, Jichi Medical University, ²Division of Nephrology, Department of Internal Medicine,Jichi Medical University, ³Department of Medical Physiology,Meiji Pharmaceutical University

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Overview

This protocol outlines the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. It is designed for studying microRNA expression profiles under various pathological conditions.

Key Study Components

Area of Science

Neuroscience
Biology
MicroRNA research

Background

MicroRNAs play crucial roles in gene regulation.
Understanding their expression in pathological conditions is vital.
Rat peritoneal membrane serves as a model for studying these expressions.
Quantitative real-time PCR is a reliable method for detection.

Purpose of Study

To purify and detect miRNAs in the rat peritoneal membrane.
To analyze microRNA expression profiles in various conditions.
To provide a detailed protocol for researchers in the field.

Methods Used

Inhalational anesthesia with 4% isoflurane.
Preparation of the abdominal area with 70% ethanol.
Surgical incision to access the peritoneal membrane.
Use of scissors and forceps for membrane extraction.

Main Results

Successful purification of miRNAs from the peritoneal membrane.
Quantitative detection of microRNA expression achieved.
Protocol validated for use in various pathological studies.
Potential applications in understanding disease mechanisms.

Conclusions

The protocol is effective for studying microRNA in rat models.
It provides insights into the role of microRNAs in pathology.
Future research can build on this methodology for broader applications.

Frequently Asked Questions

What is the significance of microRNA in research?

MicroRNAs are crucial for gene regulation and understanding their expression can reveal insights into various diseases.

How is the peritoneal membrane accessed in this protocol?

The peritoneal membrane is accessed through a surgical incision after anesthesia and preparation of the abdominal area.

What method is used for detecting microRNA expression?

Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) is used for detection.

Can this protocol be adapted for other species?

While this protocol is designed for rats, adaptations may be possible for other species with appropriate modifications.

What are the potential applications of this research?

This research can help in understanding the role of microRNAs in various pathological conditions and diseases.

Is prior experience in surgery required for this protocol?

Yes, familiarity with surgical techniques is recommended for safely performing the procedure.

Qui presentiamo un protocollo per la rilevazione dell'espressione di microRNA nella membrana peritoneale del ratto utilizzando la reazione a catena di polimerasi reattiva-trasversale in tempo reale. Questo metodo è adatto allo studio del profilo di espressione di microRNA nella membrana peritoneale del ratto in diverse condizioni patologiche.

L'obiettivo generale di questa procedura è quello di purificare e rilevare con successo i miRNA nella membrana peritoneale del ratto. Per iniziare questa procedura, dopo anestesia inalatoria con isoflurano al 4%. Spruzzare la pelle addominale del ratto con etanolo al 70% e montarlo sul foglio di sughero, sul dorso.

Quindi praticare un'incisione longitudinale nella pelle addominale, nei muscoli e nella membrana peritoneale dell'animale usando forbici e pinze. Prelevare la membrana peritoneale usando la pinza e praticare incisioni orizzontali sulla sua parte superiore lungo l'osso costale più basso sotto il diaframma. E sul lato inferiore sulla regione laterale usando le forbici chirurgiche.

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