Fecal (micro) RNA Isolation

Fyonn H. Dhang; Howard L. Weiner; Shirong Liu

doi:10.3791/61908

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Fecal (micro) RNA Isolation

Fekal (mikro) RNA İzolasyonu

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05:35 min

October 28, 2020

DOI: 10.3791/61908-v

Fyonn H. Dhang^1,2, Howard L. Weiner^1,2, Shirong Liu^1,2

¹Ann Romney Center for Neurologic Diseases, Department of Neurology, Partners Multiple Sclerosis Center,Brigham and Women's Hospital and Harvard Medical School, ²Evergrande Center for Immunologic Diseases,Harvard Medical School and Brigham and Women's Hospital

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Overview

This protocol presents a straightforward method for isolating high-quality RNA from fecal samples, minimizing contamination from living microbes. It is designed for both animal and human subjects, ensuring optimized yield and purity for downstream applications.

Key Study Components

Area of Science

Neuroscience
Microbiology
Molecular Biology

Background

Isolation of RNA from fecal samples is crucial for studying microRNAs.
Traditional methods may lead to contamination from microbial RNA.
High-quality RNA is essential for accurate downstream assays.
This protocol adapts a commercial miRNA isolation kit for improved results.

Purpose of Study

To provide a reliable method for RNA extraction from feces.
To enhance the quality and quantity of RNA isolates.
To facilitate research on microRNAs in various biological contexts.

Methods Used

Resuspension of fecal samples in sterile 1X DPBS.
Use of a two-milliliter microcentrifuge tube for processing.
Adaptation of a commercial miRNA isolation kit.
Minimization of RNA contamination from living microbes.

Main Results

Successful isolation of high-quality total RNA from fecal samples.
Optimized protocol yields RNA suitable for sequencing and RT-PCR.
Demonstrated effectiveness in reducing microbial RNA contamination.
Protocol is simple and straightforward for researchers.

Conclusions

This protocol provides a reliable method for RNA extraction from fecal samples.
High-quality RNA is crucial for downstream applications.
Adaptations to commercial kits can enhance RNA yield and purity.

Frequently Asked Questions

What is the main advantage of this RNA isolation protocol?

The protocol minimizes contamination from living microbes, ensuring high-quality RNA for downstream assays.

Can this protocol be used for both human and animal fecal samples?

Yes, the protocol is designed for isolating RNA from both human and animal fecal samples.

What downstream applications can the isolated RNA be used for?

The RNA isolates are suitable for sequencing, micro-array, and RT-PCR assays.

Is the protocol complicated to follow?

No, the protocol is simple and straightforward, making it accessible for researchers.

What is the role of the binding buffer in RNA isolation?

In this protocol, the binding buffer is not used to minimize contamination from microbial RNA.

Bu protokol, hayvan ve insan deneklerin dışkı örneklerinden yüksek kaliteli toplam RNA'yı izole eder. Ticari bir miRNA izolasyon kiti, saf RNA'yı optimize edilmiş miktar ve kalitede izole etmek için önemli bir adaptasyonla kullanılır. RNA izolatları, dizileme, mikro dizi ve RT-PCR gibi çoğu aşağı akış RNA testi için iyidir.

MikroRNA'ları incelemek için dışkı örneklerinden RNA'yı izole etmek için bir protokol sunuyoruz. Bu protokol, RNA'yı dışkıdan yüksek kalite ve miktarda izole etmek için kullanılabilir. Canlı mikroplardan gelen RNA'ları en aza indirir.

Bunu vurgulamak önemlidir ve bu protokolde bağlayıcı tampon kullanılmamaktadır. Bu protokol basit ve anlaşılır. İki mililitrelik bir mikrosantrifüj tüpünde 600 mikrolitre steril 1X DPBS'de 25 ila 100 miligram dışkı örneğini yeniden süspanse ederek başlayın.

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