Name: from mrna to cdna for gene studies
Uploaded: 2026-08-26T08:20:09.925Z
Duration: 2 min 25 s
Channel: JoVE

Almost every cell in the body has the same DNA.However, different cell types, such as neurons and muscle cells, express different sets of genes. These genes are transcribed into different messenger RNAs, or mRNAs, in each cell.In the laboratory, mRNA can be used as a template to synthesize complementary DNA, or cDNA. Since cDNA is synthesized from cellular mRNA, it represents the genes expressed by the cell.One common method begins with extracting RNA from cells.Following cell lysis and removal of cellular debris, the mRNA is isolated from other types of RNA, such as ribosomal RNA and transfer RNA. This is commonly done by adding the sample to a column containing beads with short stretches of thymine nucleotides attached.These thymine stretches bind to the poly-A tail, a chain of adenine nucleotides found at the 3-prime end of eukaryotic mRNA, through complementary base pairing.Other types of RNA lack poly-A tails, so they do not bind to the column and are washed away.An elution buffer with a low salt concentration is then added. This disrupts the interaction between the poly-A tail and the thymine stretches, releasing the bound mRNA from the beads.After the mRNA is isolated, an oligo-dT primer binds to the poly-A tail and provides a starting point for reverse transcriptase enzymes to synthesize a single-stranded cDNA copy of the mRNA.RNase enzymes are then added to degrade the RNA strand of the RNA-cDNA hybrid.Next, DNA polymerase enzymes synthesize a complementary DNA strand, producing double-stranded cDNA.This cDNA can then be inserted into plasmids or viral vectors for molecular biology research.

complementary dna

Complementary DNA

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA&#x2014;unlike genomic DNA&#x2014;also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene.cDNA SynthesiscDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types&#x2014;transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail&#x2014;a string of adenine nucleotides&#x2014;added to its 3&#x2019; end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away.Next, reverse transcriptase&#x2014;a DNA polymerase enzyme from retroviruses&#x2014;is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3&#x2019; end of a chain, a poly(T) primer is added to bind to the poly(A) tail to provide a starting point for cDNA synthesis. The cDNA strand ends in a hairpin loop. The RNA is then degraded&#x2014;commonly with alkali treatment or RNase enzymes&#x2014;leaving the single-stranded cDNA intact.A second DNA strand complementary to the cDNA is then synthesized by DNA polymerase&#x2014;often using the hairpin loop of the first cDNA strand or a nicked piece of the mRNA as a primer.The resulting double-stranded cDNA can be inserted into bacterial or viral vectors and cloned using standard molecular biology techniques. A cDNA library&#x2014;representing all the mRNAs in the cells or tissue of interest&#x2014;can also be constructed for additional research.

Overview

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to ...

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from mrna to cdna for gene studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples.

cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help researchers determine the amino acid sequence of the peptide encoded by a gene, unlike genomic DNA.

To make cDNA, scientists often start by extracting total RNA from cells. They then isolate mRNA from the more abundant transfer RNA (tRNA) and ribosomal RNA (rRNA). Mature eukaryotic mRNA has a poly(A) tail, which is a chain of adenine nucleotides added to the 3' end. Other RNA types do not have this feature.

Because of the poly(A) tail, oligo-dT molecules can be attached to a column or magnetic beads to capture mRNA by base pairing. The other RNA molecules are washed away. Next, reverse transcriptase, a DNA polymerase enzyme from retroviruses, builds cDNA from the mRNA template. A poly(T) primer binds to the poly(A) tail and gives the enzyme a starting point, since it adds nucleotides only to the 3' end of a chain.

The first cDNA strand ends in a hairpin loop. The RNA is then degraded, often with alkali treatment or RNase enzymes, and the single-stranded cDNA remains. A second DNA strand is then synthesized by DNA polymerase. It may use the hairpin loop of the first strand or a nicked piece of mRNA as a primer. The final double-stranded cDNA can be inserted into bacterial or viral vectors and cloned with standard molecular biology methods. Researchers can also build a cDNA library that represents the mRNAs found in a specific cell or tissue sample.

From mRNA to cDNA for Gene Studies

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Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

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key terms and concepts

Learn how scientists isolate mRNA, use reverse transcription, and make cDNA for gene expression studies, cloning, and cDNA libraries in cells and tissues.

From mRNA to cDNA for Gene Studies | JoVE K12

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From mRNA to cDNA for Gene Studies

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