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Transcriptome

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The transcriptome is the complete set of all RNA molecules, or transcripts, found within a cell or a population of cells. It includes functional RNA molecules that help the cell operate. It also includes other transcripts that arise from non-functional regions, such as pseudogenes or virus fragments. Scientists aim to distinguish between functional transcripts and what is often called "junk RNA."

Metabolomics schema
Metabolomics schema

While the genome is the static DNA sequence, the transcriptome is dynamic. It changes based on cell type, developmental stage, and environmental stimuli. This makes the transcriptome a reflection of a cell's active gene expression state.

RNA molecules are created through a molecular process called transcription. This process begins when an enzyme called RNA polymerase recognizes a promoter sequence. The promoter is located near the transcription start site and defines where a gene begins. Transcription factors often help regulate and mediate this initiation. Once the enzyme attaches to the template DNA strand, it catalyzes the addition of ribonucleotides. These are added to the 3' end of the growing RNA sequence. The resulting RNA strand is complementary to the DNA strand it originated from. The process concludes when the enzyme reaches a terminator site, which is often identified by specific termination sequences.

There are many different types of RNA transcripts within a transcriptome. Ribosomal RNA, or rRNA, is usually the most abundant type. Long non-coding RNA (lncRNA) refers to transcripts longer than 200 nucleotides. These make up the largest fraction of the non-coding transcriptome after introns. Other small RNAs include microRNA (miRNA), which are 19 to 24 nucleotides long. MicroRNAs can regulate the expression of mRNA through a process called RNA interference. There are also Piwi-interacting RNAs (piRNA) that are 24 to 31 nucleotides long. These interact with Piwi proteins to target and cleave transposons.

In many organisms, the transcriptome contains transcripts that do not come from known genes. In mammals, known genes only account for 40% to 50% of the genome. Because of this, identified transcripts often map to a much larger fraction of the genome than the genes themselves. This suggests the presence of spurious transcription. Some of these transcripts come from transcribed pseudogenes or degenerative transposons and viruses. Many scientists assume that if a transcript is not assigned to a known gene, it is junk RNA until proven functional. This is especially common in eukaryotes with very large genomes.

The history of studying the transcriptome involves several major milestones. The first study to present a cDNA library of silk moth mRNA was published in 1979. A seminal study in 1997 investigated the transcriptome of the organism S. cerevisiae. This study used serial analysis of gene expression, or SAGE, to describe 60,633 transcripts. The field grew as automated DNA sequencing became available during the 1980s. In the 1990s, researchers used expressed sequence tag sequencing to identify gene fragments. As computational power and high-throughput technologies increased, analyzing enormous amounts of data became much easier.

Scientists use different methods to construct and analyze a transcriptome. One older method involves DNA microarrays, also known as DNA chips.

Affymetrix-microarray
Affymetrix-microarray

Microarrays consist of thin glass layers with spots containing known DNA sequences called probes. Researchers convert mRNA into cDNA and mark it with fluorescent colors, such as red or green. When the cDNA hybridizes to the chip, a laser scans the fluorescence to measure gene expression. However, microarrays cannot provide information about unknown genes. Most researchers have now moved toward next-generation sequencing techniques.

RNA sequencing is a modern technology that allows for both qualitative and quantitative analysis. It requires only a small amount of RNA and does not require prior knowledge of the genome. This allows scientists to discover entirely new transcripts. The process involves RNA purification, followed by the synthesis of an RNA or cDNA library. Scientists often use UV spectrometry to measure RNA quality by looking at an absorbance peak at 260 nm. They may also use the RNA Integrity Number (RIN) to compare the ratio of 28S RNA to 18S RNA.

Affymetrix-microarray
Affymetrix-microarray

Recent advancements even allow for single-cell transcriptomics, which studies the transcriptome of individual cells, including bacteria.

Understanding the transcriptome is essential for modern molecular biology. It helps researchers catalogue all species of transcripts and determine their transcriptional structure. This includes identifying start sites, splicing patterns, and post-transcriptional modifications. By studying transcriptomics, scientists can quantify how expression levels change during development or under different conditions. This field connects the static information in the genome to the actual biological functions happening in a living system. It provides a window into how life responds to the world around it.

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