Biology
A researcher is studying the transcriptome in 2 different organisms. What type of primer should she
use for generating her cDNA libraries: (2 points)
a. to compare the transcriptome of a bacteria treated with sub-therapeutic level of an antibiotic versus a
bacteria treated with a vehicle control?
b. to compare the transcriptome at multiple time points during the early development of a mouse
embryo?
three type of primers for DNA synthesis are oligo dt, random short primers and sequence-specific
primer.
Answers
Answer:
Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, we review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions.
INTRODUCTION
RNA molecules are essential components of all living cells. Understanding the identity and abundance of each RNA molecule in a given cell under a specific condition is the ultimate goal of RNA research. Much of what we know about RNA comes from studies using biochemical methods, where a small number of specific molecules are analyzed. High-throughput approaches that enable interrogation of RNA sequences on a large scale emerged in the early 1990s. The expressed sequence tag (EST) method developed by Adams et al. examines gene expression by partially sequencing complementary DNA (cDNA) clones, revealing both the sequence and the abundance of corresponding RNAs.1 EST data played a pivotal role in identification of new genes in genomes in the 1990s. However, the high sequencing cost of the method limited its use in expression analysis, and the data is largely believed to be semi-quantitative. The Serial Analysis of Gene Expression (SAGE) method developed by Velculescu et al. significantly cut down the cost of expression analysis on a per gene basis,2 thanks to sequencing only a short tag region per cDNA (15 bp for the short SAGE method and 21 bp for the long SAGE method). However, the emergence of DNA microarray technology in the mid-1990s superseded EST and SAGE methods for gene expression analysis, largely due to its much better affordability for large scale studies.3,4 DNA microarray analysis of gene expression is based on hybridization of fluorescently labeled targets that are derived from transcripts to probes that are attached to a solid surface through printing or in situ synthesis. However, while the method enables interrogation of transcripts genome-wide, the requirement that a priori sequence information or reference genomes/transcriptomes be available for designing the microarray probes limited the development and application of this technology in discovery applications. In addition, cross-hybridization and background signals often lead to low specificity or low sensitivity for some genes.
The first decade of this millennium witnessed the advent of massive parallel sequencing, also known as deep sequencing or Next Generation Sequencing (NGS). Lauded as revolutionary in biology and medicine for its ability to acquire an unprecedented amount of data in a short time, deep sequencing quickly transformed RNA research. RNA-Seq is now the method of choice to study gene expression and identify novel RNA species. Compared to DNA microarray-based methods, RNA-Seq offers less background noise and a greater dynamic range for detection. Most importantly, RNA-Seq directly reveals sequence identity, crucial for analysis of unknown genes and novel transcript isoforms. Several different technologies have been developed for RNA-Seq.5–9 Here we review general aspects of RNA-Seq and applications of RNA-Seq to study specific problems. We discuss issues and remedies related to bias and sensitivity in these methods, two paramount concerns in an RNA-Seq experiment. We also discuss specialized methods that investigate aspects of RNA biogenesis and metabolism.
GENERAL ASPECTS OF RNA-Seq
While direct sequencing of RNA molecules is possible,10 most RNA-Seq experiments are carried out on instruments that sequence DNA molecules due to the technical maturity of commercial instruments designed for DNA-based sequencing. Therefore, cDNA library preparation from RNA is a required step for RNA-Seq. Each cDNA in an RNA-Seq library is composed of a cDNA insert of certain size flanked by adapter sequences, as required for amplification and sequencing on a specific platform. The cDNA library preparation method varies depending on the RNA species under investigation, which can differ in size, sequence, structural features and abundance. Major considerations include (1) how to capture RNA molecules of interest; (2) how to convert RNA to double-stranded cDNAs with defined size ranges; and (3) how to place adapter sequences on the cDNA ends for amplification and sequencing. These are discussed in the following sections.
please like and follow