In situ RNA sequencing

Biologists have invented subcellular RNA sequencing

<url>Biologists led by renowned geneticist George Church have created an in situ RNA sequencing method that allows you to map the expression of thousands of genes on a scale from the subcellular level to the whole brain or embryo.

Red highlights the "working" RNA molecules in the mouse brain, green – in the mouse embryo
(Photo: Wyss Institute – VM)

The description of the method is published in Science (Je Hyuk Lee et al., Highly Multiplexed Subcellular RNA Sequencing in Situ), a brief description of it can be read in the press release of the Wyss Institute A bird's eye view of cellular RNAs.

The method is called FISSEQ (fluorescent RNA sequencing in situ). The Latin expression in situ biologists denote those methods that give spatial information – the data of which are tied to a specific point of the cell or organism. In this case, the sequencing of FISSEQ allows you to determine the sequences of RNA molecules that are located in different parts of the cell. Sequencing takes place with a high resolution resembling optical microscopy.

The procedure looks like this. First, the sample is fixed and cut into thin layers. Then the membranes are removed from the cells, while RNA, DNA and proteins remain fixed in the matrix (this method of "enlightenment" was invented recently in order to obtain a transparent brain). The RNA is translated by reverse transcriptase into the corresponding DNA copy, which is then repeatedly amplified. As a result, a cluster of identical fixed molecules ("nanosheet") is formed from one RNA molecule. It does not represent the entire source RNA, but only a fragment of it, but this fragment is sufficient for reliable identification of the entire molecule.

In situ sequencing. Each point is a fluorescent signal from a "nanoshark" of a specific RNA
Frame: Wyss Institute/Nature

Then, already known pyrosequencing methods are used: labeled bases are added to the sample, and during the synthesis of new DNA copies, different signals light up at different points in the sample.

Before the invention of FISSEQ, biologists could either determine the spatial distribution of several RNAs (in situ FISH tagging method), or find out the sequence of all RNAs, but lose spatial information (sequencing). The new method combines both possibilities, which is likely to lead to the emergence of a new class of research in embryology and brain research.

Portal "Eternal youth" http://vechnayamolodost.ru03.03.2014