Intracellular RNA-Seq

from Genetic Engineering News by Anton Simeonov, Ph.D.

This literature review highlights a study led by George Church describing FISSEQ, or fluorescent in situ RNA sequencing.

Methods such as fluorescence in situ hybridization (FISH) allow gene expression to be observed at the tissue and cellular level; however, only a limited number of genes can be monitored in this manner, making transcriptome-wide studies impractical. George Church’s group* is presenting the further development of their original approach called fluorescent in situ sequencing (FISSEQ) to incorporate a spatially structured sequencing library and an imaging method capable of resolving the amplicons (see Figure 1).In fixed cells, RNA was reverse transcribed with tagged random hexamers to produce cDNA amplicons. Aminoallyl deoxyuridine 5-triphosphate (dUTP) was incorporated during reverse transcription and after the cDNA fragments were circularized before rolling circle amplification (RCA), an amine-reactive linker was used to cross-link the RCA amplicons containing aminoallyl dUTP. The team generated RNA sequencing libraries in different cell types, tissue sections, and whole-mount embryos for three-dimensional (3D) visualization that spanned multiple resolution scales (read more…)