Background: RNA sequencing (method 1)

The goal of the method
RNA sequencing is a method that is used a lot in molecular biology laboratories around the world. The method is used to measure the expression of genes. All cells in a certain organism have the same genes. Differences in the expression of those genes between the cells results in different cells being able to perform different functions: an immune cell can combat pathogens, a muscle cell can contract, a cell in a plant leaf can perform photosynthesis. Therefore, studying gene expression can learn us more about the developmental processes underlying the differentiation of the cells and the formation of multicelllular organisms like us humans. In addition, changes in gene expression allow a cell – and thus also multicellular organisms –  to respond to the environment. Knowing which genes are expressed in response to certain stresses, such as heart failure in humans, or drought in plants, can give us an idea of how cells and organisms deal with those stresses. Ultimately, this can aid in for example developing treatments to prevent heart failure in humans or breeding programs to increase tolerance to drought in crop species.

The method itself
RNA sequencing measures gene expression by measuring the amount of RNA present. As explained here, RNA is the intermediate in the process of using the DNA code as a ‘building plan’ to form a protein. The more RNA from a certain gene is detected, the more active the gene was. Once the order of letters in a string of RNA, in other words the sequence of a piece of RNA, is known, it is easy to determine from which gene it originated. This is easy because the sequence of a piece of RNA corresponds to certain letters of the DNA sequence it was made from: an A in the RNA is a T in the DNA, a G in the RNA is a C in the DNA, etc. Thus, once the sequence of a piece of RNA is known, one can deduce the sequence of the DNA it was made from. With databases on the internet, the gene corresponding to this DNA sequence can be determined.

To measure the amount of RNA present, the RNA is first converted to cDNA (complementary DNA) by the researcher. Thus, synthetic A, T, C and Gs are combined with the mRNA pieces, in addition to ‘reverse transcriptase’: a protein that can bind DNA nucleotides one by one to an mRNA molecule to create a complementary piece of DNA: the cDNA. This is necessary, because the sequencer, the machine that reads the sequences, cannot read RNA. The cDNA samples are then sent off to a sequencing facility, where the sequencer ‘reads’ all the pieces of cDNA. In other words, it reads the order of A, T, C and Gs of all the pieces of the cDNA present. This information is put into a huge file, containing all the sequences, the so-called reads, that the sequencer read. This file is returned back to the researcher, who can use a computer program to find out from which DNA the RNA-turned-into-cDNA pieces originated.


In the above-shown case, the red lines represent the RNA sequences of which the cDNA pieces were read by the sequencer. The black line represents the DNA of the studied organism. With a computer program the reads have been aligned to the genome based on their sequence . Finally, by looking up which parts of the DNA correspond to genes – this information is available online for most organisms -, the researcher knows which genes are expressed. In this case, gene 3 is expressed a lot, whereas gene 4 is not expressed at all.

Use of the method
Generally, the gene expression of not one, but several samples is determined in an RNA sequencing. A healthy heart of a newborn and a healthy heart of an adult, for example, or a diseased heart and a healthy heart. By comparing gene expression in the two (or more) conditions, one can learn about developmental processes and/or stress responses.

RNA sequencing is used in many different fields of biology, ranging from cancer biology to microbiology. On this website several papers are discussed using this method: this paper on light perception in plant roots and this paper on heart development.

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