Question

RNA-Seq is a next-generation sequencing method used to quantitatively profile the cellular transcriptome. Researchers use RNA-Seq to compare the expression of genes under different environmental conditions or between different types of cells. There are three general steps in an RNA-Seq workflow: 1\. Generate a cDNA library from cellular RNA. 2\. Add oligonucleotide adapters to the fragments of the cDNA library. 3\. Use next-generation sequencing to identify transcriptionally active genes from the cDNA library. What is the role of the enzyme reverse transcriptase in an RNA-Seq workflow?

Short answer

Reverse transcriptase converts RNA into cDNA in RNA-Seq.

Step-by-step solution

Understanding Reverse Transcriptase

Reverse transcriptase is an enzyme that synthesizes complementary DNA (cDNA) from an RNA template. It is crucial for allowing the conversion of RNA sequences into stable DNA sequences which can then be further manipulated or sequenced.

Identifying the Role in RNA-Seq Workflow

In the RNA-Seq workflow, reverse transcriptase is specifically used in the first step, where a cDNA library is generated from cellular RNA. Without the action of reverse transcriptase, the RNA cannot be converted into cDNA, which is essential for subsequent sequencing steps.

Creating cDNA Library

During the creation of a cDNA library, reverse transcriptase acts on the extracted RNA to create a complementary DNA version. This cDNA serves as a stable template for downstream applications, like sequencing, providing a 'snapshot' of the gene expression profiles of the cell's transcriptome.

Key concepts

Reverse Transcriptase

Reverse transcriptase is a fascinating enzyme that plays a pivotal role in molecular biology, particularly in RNA-Seq workflows. This enzyme has the unique ability to transcribe RNA into complementary DNA (cDNA), essentially creating a genetic "mirror image" of the RNA sequence. This process is vital because RNA is inherently unstable and can degrade easily.

Here are some key points about reverse transcriptase:

• It was first discovered in retroviruses, which use it to convert their RNA genomes into DNA to integrate into the host's genome.
• In the laboratory, reverse transcriptase is widely used to create cDNA libraries, which are crucial for studying gene expression.
• It bridges the gap between RNA and DNA, allowing scientists to handle, store, and sequence genetic information more efficiently.

The action of reverse transcriptase marks the first step in the RNA-Seq process. Its role is to convert RNA into cDNA, preserving the information in a more stable and manageable form, setting the stage for further analysis like sequencing.

cDNA Library

A cDNA library is essentially a collection of cDNA sequences that represent the transcripts present in a sample at a given time. By "snapshotting" the gene expression, researchers can understand which genes are active, their expression levels, and how various conditions or treatments impact them.

The main steps involved in creating a cDNA library include:

• Extracting the total RNA from the cells or tissue of interest.
• Using reverse transcriptase to produce a complementary DNA strand from the RNA template.
• Linking oligonucleotide adapters to the cDNA fragments to facilitate sequencing.

This library serves as a crucial intermediate product in RNA-Seq. It captures the transcriptome's complexity and diversity and acts as a stable template for downstream sequencing. In other words, it lets researchers explore the universe of expressed genes in great detail, paving the way for understanding gene function and regulation.

Next-Generation Sequencing

Next-generation sequencing (NGS) has revolutionized genomics by allowing researchers to sequence entire genomes or isolated transcriptomes rapidly and cost-effectively. In the context of RNA-Seq, NGS refers to the technology used to read the cDNA sequences from the cDNA library.

Here are some attributes of NGS:

• It provides high-throughput data, meaning it can process millions of sequences simultaneously.
• NGS generates massive amounts of data, offering insights into gene expression, variations, and mutations.
• The sequencing platforms are versatile and can be adjusted for varying read lengths and depths, increasing experimental flexibility.

The implementation of NGS in RNA-Seq allows scientists to identify which genes are switched on or off in a cell under specific conditions. This pushes the boundaries of biological research by enabling comprehensive analyses of the genetic underpinnings of disease, development, and environmental responses.