Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

Lim Chee Liew; Yan Wang; Marta Peirats-Llobet; Oliver Berkowitz; James Whelan; Mathew  G. Lewsey

doi:10.3791/61517

레이저 캡처 미세 절 RNA-시퀀싱 용 공간 및 측두 조직 -특정 유전자 발현 분석

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Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

레이저 캡처 미세 절 RNA-시퀀싱 용 공간 및 측두 조직 -특정 유전자 발현 분석

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08:33 min

August 5, 2020

DOI: 10.3791/61517-v

Lim Chee Liew^1,2, Yan Wang^1,2, Marta Peirats-Llobet¹, Oliver Berkowitz^1,2,3, James Whelan^1,2,3, Mathew G. Lewsey^1,3

¹Department of Animal, Plant and Soil Science, AgriBio Building,La Trobe University, ²Australian Research Council Centre of Excellence in Plant Energy Biology,La Trobe University, ³Australian Research Council Research Hub for Medicinal Agriculture, AgriBio Building,La Trobe University

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Overview

This study presents a protocol for Laser-Capture Microdissection (LCM) coupled with RNA sequencing to analyze spatial and temporal transcriptomes in plant cells. The technique allows for precise isolation of cells within their native tissue context, facilitating detailed gene expression studies.

Key Study Components

Research Area

Plant biology
Molecular biology
Transcriptomics

Background

Importance of tissue-specific gene expression analysis
Need for contamination-free techniques in sample preparation
Benefits of RNA sequencing in understanding plant biology

Methods Used

Laser-Capture Microdissection (LCM)
Plant cells, specifically from barley seeds
RNA extraction and sequencing

Main Results

Successfully isolated specific cells for RNA analysis
Identified gene expression patterns over 48 hours during germination
Demonstrated the effectiveness of LCM for high-quality RNA samples

Conclusions

The protocol enhances understanding of spatial and temporal gene expression in plants.
It provides a valuable method for researchers studying developmental biology and genetics.

Frequently Asked Questions

What is Laser-Capture Microdissection?

Laser-Capture Microdissection is a technique used to isolate specific cells from tissue sections with minimal contamination.

How does RNA sequencing complement LCM?

RNA sequencing allows for the analysis of gene expression profiles from the isolated cells, providing insights into their function and behavior.

What types of samples can be processed with this protocol?

While optimized for plant cells, the protocol can be adapted for various tissue types that can be histologically identified.

Why is sample preparation critical in this process?

Proper fixation and embedding are essential to maintain tissue integrity and ensure successful dissection and RNA extraction.

What are the key findings of this research?

The research highlights distinct gene expression patterns in various plant tissues during germination, revealing insights into developmental processes.

How long does the protocol take?

The entire process, including sample preparation and RNA sequencing, requires careful management of time but typically spans several days.

What are the implications of this research?

The study contributes to our understanding of gene regulation in plants, which is crucial for fields like agriculture and biotechnology.

여기에 제시된 것은 식물 조직의 레이저 포획 미세 절(LCM)을 위한 프로토콜입니다. LCM은 오염없는 방식으로 조직의 영역을 격리하기위한 현미경 기술입니다. 절차는 조직 고정, 파라핀 포함, 단면, LCM 및 RNA 추출을 포함한다. RNA는 다운스트림 조직 특이적, 전사의 현세적으로 해결된 분석에서 사용된다.

이 프로토콜은 소량의 생물학적 물질을 사용하여 이해 분야의 특정 세포로부터 공간 및 측두전사를 얻기 위해 RNA-시퀀싱과 결합된 레이저 포획 미세 절위를 사용합니다. 레이저 기술의 주요 장점은 정상적인 조직 컨텍스트 내에서 세포의 직접 시각화를 용이하게하여 이산 세포가 접촉없는 방식으로 정확하게 분리 될 수 있다는 것입니다. 이 프로토콜은 식물 세포의 격리에 최적화되어 있지만, 그것은 stologically 식별 될 수있는 대부분의 세포에 적용 할 수있다.

좋은 샘플 준비는 중요합니다. 따라서, 이 기술을 처음으로 수행하는 사람은, 조직 고정 및 포함의 소품 최적화는 레이저 캡처 마이크로절 전에 중요하다. 조직 샘플을 수집하기 전에, 수확할 종 및 조직 유형에 적합한 고정을 준비한다.

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