Difference Between Microarray and RNA Sequencing

Key Difference – Microarray vs RNA Sequencing

Transcriptome represents the whole content ofRNApresent in a cell includingmRNA,rRNA,tRNA, degraded RNA, and, nondegraded RNA. Profiling transcriptome is an important process in order to understand the cell insights. There are several advanced methods for transcriptome profiling. Microarray and RNA sequencing are two types of technologies developed to analyze transcriptome. The key difference between microarray and RNA sequencing is thatmicroarray is based on the hybridization potential of predesigned labeled probes with target cDNA sequences while RNA sequencing is based on the direct sequencing of cDNA strands by advanced sequencing techniques such asNGS.Microarray is performed with the prior knowledge about the sequences and RNA sequencing is performed without the prior knowledge about sequences.

CONTENTS
1.Overview and Key Difference
2.What is Microarray
3.What is RNA Sequencing
4.Side by Side Comparison – Microarray vs RNA Sequencing
5.Summary

What is Microarray?

Microarray is a robust, reliable and high throughput method used for transcriptome profiling by scientists. It is the most popular approach for transcript analysis. It is a low-cost method, which depends on the hybridization probes.

The technique starts with extraction of mRNA from the sample and the construction ofcDNA libraryfrom total RNA. Then it is mixed with fluorescently labeled predesignedprobeson a solid surface (spot matrix). Complementary sequences hybridize with the labeled probes in the microarray. Then microarray is washed and screened, and the image is quantified. Gathered data should be analyzed to get the relative expression profiles.

The intensity of the microarray probes is assumed to be proportional to the quantity of transcripts in the sample. However, the accuracy of the technique depends on the designed probes, prior knowledge of the sequence and the affinity of probes forhybridization. Hence microarray technology has limitations. Microarray technique cannot be performed with low abundance transcripts. It fails to differentiate isoforms and identify genetic variants. Since this method depends on hybridization of probes, some problems related to hybridization such as cross-hybridization, nonspecific hybridization etc. occurs in microarray technique.

What is RNA Sequencing?

RNA shotgun sequencing (RNA seq) is a recently developed whole transcriptome sequencing technique. It’s a rapid and high throughput method of transcriptome profiling. It directly quantifies the expression ofgenes和结果深陷发票estigation of the transcriptome. RNA seq does not depend on predesigned probes or prior knowledge of the sequences. Therefore, RNA seq method has high sensitivity and capability of detecting novel genes and genetic variants.

RNA sequencing method is performed via several steps. Total RNA of the cell must be isolated and fragmented. Then, using reverse transcriptase, a cDNA library must be prepared. EachcDNAstrand must be ligated with adaptors. Then the ligated fragments must be amplified and purified. Finally using a NGS method, sequencing of the cDNA must be performed.

What is the difference between Microarray and RNA sequencing?

Microarray vs RNA Sequencing
Microarray is a robust, reliable, high throughput method.	RNA sequencing is an accurate and high-throughput method.
Cost
This is a low-cost method.	This is an expensive method.
Analysis of a Large Number of Samples
This facilitates analyzing a large number of samples simultaneously.	This facilitates analyzing a large number of samples.
Data Analysis
Data analysis is complex.	More data is generated in this method; hence, the process is more complex.
Prior Knowledge of Sequences
This method is based on hybridization probes, so prior knowledge of sequences in required.	This method does not depend on the prior sequence knowledge.
Structural Variations and Novel Genes
This method cannot detect structural variations and novel genes.	This method can detect structural variations such as gene fusing, alternative splicing, and novel genes.
Sensitivity
This cannot detect differences in expression of isoforms, so this has limited sensitivity.	高灵敏度。
Outcome
This can only result in relative expression levels. This does not give absolute quantification of gene expression.	It gives absolute and relative expression levels.
Data Reanalysis
This needs to be rerun in order to reanalyze.	Sequencing data can be reanalyzed.
Need for Specific Personnel and Infrastructure
Specific infrastructure and personnel are not required for microarray.	Specific infrastructure and personnel required by RNA sequencing.
Technical Issues
Microarray technique has technical issues such as cross-hybridization, nonspecific hybridization, limited detection rate of individual probes, etc.	RNA seq technique avoids technical issues such as cross-hybridization, nonspecific hybridization, limited detection rate of individual probes, etc.
Biases
这是一个有偏见的方法it depends on hybridization.	Bias is low compared to microarray.

Summary – Microarray vs RNA Sequencing

Microarray and RNA sequencing methods are high throughput platforms developed for transcriptome profiling. Both methods produce results which are highly correlated to gene expression profiles. However, RNA sequencing has advantages over microarray for gene expression analysis. RNA sequencing is a more sensitive method for the detection of low abundance transcripts than microarray. RNA sequencing also enables the differentiation between isoforms and identification of gene variants. However, microarray is the common choice of most researchers since RNA sequencing is a new and expensive technique with data storing challenges and complex data analysis.

References:
1.Wang, Zhong, Mark Gerstein, and Michael Snyder. “RNA-Seq: a revolutionary tool for transcriptomics.” Nature reviews. Genetics. U.S. National Library of Medicine, Jan. 2009. Web. 14 Mar. 2017
2.Rogler, Charles E., Tatyana Tchaikovskaya, Raquel Norel, Aldo Massimi, Christopher Plescia, Eugeny Rubashevsky, Paul Siebert, and Leslie E. Rogler. “RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples.” Nucleic Acids Research. Oxford University Press, 01 Jan. 2004. Web. 15 Mar. 2017
3.Zhao, Shanrong, Wai-Ping Fung-Leung, Anton Bittner, Karen Ngo, and Xuejun Liu. “Comparison of RNA-Seq and Microarray in Transcriptome Profiling of Activated T Cells.” PLOS ONE. Public Library of Science, Jan. 2014. Web. 15 Mar. 2017

Image Courtesy:
1.“Journal.pcbi.1004393.g002”By Malachi Griffith, Jason R. Walker, Nicholas C. Spies, Benjamin J. Ainscough, Obi L. Griffith –(CC BY 2.5)viaCommons Wikimedia
2. “Microarray” By Bill Branson (Photographer) – National Cancer Institute (Public Domain) viaCommons Wikimedia