cDNA Synthesis: Overview & Applications

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cDNA Synthesis: Overview

cDNA synthesis is a process of producing complementary DNA (cDNA) by reverse transcription. The process utilizes mRNA or miRNA as a template, reverse transcriptase enzyme, and a thermostable primer, complementary to the 3’ end of the RNA template.

The synthesized cDNA products are used as a template to produce the second DNA strands using the PCR (Polymerase chain reaction) assays.

The combination of PCR and reverse transcription helps to detect the low abundance of RNA. Thus, the production of cDNA using such RNAs facilitates cloning the low copy genes. 

However, cDNA can also be generated without using PCR. This is done by using DNA Polymerase I and DNA Ligase. The workflow also requires RNase H activity and is performed when no amplification is required.

While performing cDNA synthesis, making the right choice on the primer and RTases types is essential. The three types of primers include gene-specific primers, oligo(dT) primers containing T7 promoters, and random primers (or random hexamers). 

Careful priming is important to avoid an abundance of truncated cDNA transcripts and over-representation of synthesized cDNA.

The cDNA synthesis is the first step in many in vitro life sciences and molecular biology lab workflows, such as gene cloning, qRT-PCR, creating cDNA libraries, RT-qPCR, and quantifying gene expression analysis using real-time PCR (or RT-PCR).

In this article, we will cover how cDNA is synthesized, its applications in lab workflows, and what industries use the process in their biological product development systems. 

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How Does cDNA Synthesis Work?

cDNA synthesis is also known as reverse transcription or reverse genetics, as in this process cDNA is synthesized using an mRNA template and reverse transcriptase enzyme.

The system is naturally found in a group of retroviruses, which include Murine Leukemia Virus, Lentivirus, HIV-1, immunodeficiency virus, avian myeloblastosis virus (AMV), Moloney murine leukemia virus, etc.

cDNA Synthesis

The reagents used to perform the first strand cDNA synthesis include total RNA (pretreated to remove genomic DNA and integrity checked using SDS electrophoresis or microfluidics), reverse transcriptase enzyme, DNaseI, Nuclease-Free Water, primer, SYBR green, First-Strand Synthesis System, master mix, dNTPs, Rnase inhibitor, DTT, and reaction buffer.

Generally, a recombinant M-MLV reverse transcriptase is used in cDNA synthesis. Because the enzyme has reduced ribonuclease H (or RNase H) activity and increased thermostability. Moreover, the enzyme is capable of working at temperatures up to 50°C, provides a higher yield of full-length cDNA products, and has higher specificity.

Either individual reagents are mixed or a commercially available high fidelity First Strand cDNA Synthesis Kit is used in the lab workflows.

The five steps involved in cDNA synthesis include:

  1. Sample preparation: RNA samples are prepared and purified using a suitable technique based on the goals of the experiments. 
  2. Genomic DNA removal: This is the RNA purification step. DNaseI is used to remove all the genomic DNA from the sample. 
  3. Reverse transcriptase selection: Choose reverse transcriptase based on the requirement of your results. For example, if you need to synthesize longer cDNA at a higher efficiency, use avian myeloblastosis virus (AMV) reverse transcriptase. 

However, if you need to run the process at a higher temperature and want better cDNA yield, use MMLV reverse transcriptase.

  1. Reaction mix preparation: Prepare the mix for reverse transcription by adding RNA template, dNTPs, DTT, buffer, RNAse inhibitors, and RNase-free water.

cDNA synthesis: In this step, cDNA is synthesized by primer annealing, DNA polymerization, and enzyme deactivation.

Figure: The step-wise process of cDNA synthesis.

What is cDNA Synthesis Used For?

cDNA has a spectrum of applications in cDNA library construction, gene discovery, gene cloning, and miRNA profiling.

Gene Discovery

Gene discovery is the identification of genes expressed differently in two different states, such as normal and diseased conditions. The study is performed to develop therapeutics by understanding and elucidating the organism’s genetic pathways.

cDNA microarray technique helps to achieve the goal by facilitating the profiling of complex diseases and discovering novel genes related to diseases.

Gene Cloning

Gene cloning is an experimental technique used to make identical copies of a gene or DNA segment. The desired gene segments are inserted into the plasmid using restriction enzymes and ligases. The technique is used to develop proteins (such as insulin), gene therapies, and to analyze the mutation effects of a gene. cDNA is used to clone eukaryotic genes into prokaryotes. It’s used to produce proteins in a cell that is not naturally developed by the cell on its own. Furthermore, it helps in understanding gene activities in organisms’ metabolic pathways.

Figure: The step-wise process of gene cloning.

cDNA Libraries

cDNA libraries are a collection of cloned cDNA fragments into a variety of host cells. They are broadly employed to determine the expressed portion of eukaryotic protein-coding genes. It’s used to reproduce the eukaryotic genome, express eukaryotic genes in prokaryotes,  reverse genetics, and functional cloning (identify genes based on the encoded proteins’ functions).

Figure: A schematic diagram of the formation of a cDNA library.

What Industries Use cDNA Synthesis?

cDNA synthesis is a routine lab workflow in many life sciences, biotechnology, and medical areas. 

Pharmaceutical

In pharma companies, cDNA is synthesized to understand the disease conditions and develop targeted therapies. It’s also used to develop some proteins like insulin to treat diseases and gene therapies. 

Biotechnology

In biotech, cDNA synthesis is used as a routine workflow to clone desired genes into organisms. It’s an effective way to express proteins in organisms that can’t naturally produce them. Also, the technique is useful for creating a cDNA library, storing genomic information of organisms, and gene cloning.

Molecular Biology

The cDNA synthesis is a useful tool for studying gene expression in a host of organisms. Moreover, they are used to express thousands of genes for polymorphism screening, genomic DNA clone mapping, and large-scale gene discovery.

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cDNA synthesis is a workflow used in life sciences and biotech labs to synthesize complementary DNA (cDNA). The process is facilitated by reagents like reverse transcriptase enzyme, primer, buffer, DNase, and RNase-free water.

The priming of the RNA and the type of reverse transcriptase enzyme are two essential factors that can impact the reproducibility of the cDNA. Thus, it’s necessary to choose a system based on the goals of your experiments. 

The process of cDNA synthesis has a range of applications in lab workflows, such as sequencing, qRT-PCR, gene expression studies, cDNA library preparation, gene cloning, and gene discovery studies.

Performing such high-throughput experiments needs high-grade chemicals and products and high-throughput equipment

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