Technology watch: novel ways to look at the transcriptome

26 November 2016
​Technologies for gene expression analysis have changed dramatically in the last few years. From Northern blotting and microarray-based gene expression profiling all the way to RNA-sequencing. Deciphering the transcriptome is showcasing amazing insights into how individual cells work. Two recent platforms have created quite a stir in life sciences research, as they transcend the classical bulk-transcriptome approaches.

A whole new world: 2D visualization of gene expression revealed
An exciting “next-gen transcriptomics” approach enables the visualization and quantification of gene expression in a spatial tissue-based context (Stahl et al., Science 2016). The technology, initially developed
at Karolinska Institute, is being commercialized by the company Spatial Transcriptomics. Their method, RNA sequencing in 2D, starts by attaching a tissue section onto a gridded microarray, packed with approximately 1000 different spots containing oligo-dT probes with location-specific barcodes. After histological analysis and imaging, the cells are lysed. The cellular RNA diffuses directly from the tissue section onto the array, hybridizing to the oligo-dT.

Following cDNA synthesis on-chip, the cDNAs are eluted, converted into a library and sequenced, whereby the barcodes in each sequence define the original 2-dimensional location of the RNA at high resolution. Data visualization of the tissue section image with gene expression data allows the determination of which genes
are expressed in specific tissue areas and in what quantity. The technology enables novel types of bioinformatics analyses of gene expression, which are extremely valuable in research and diagnostics.

The bead of a different sequencing technology
A second approach that goes beyond bulk population analysis for decoding cellular heterogeneity consists of single cell RNA sequencing. To isolate and sequence thousands of single cells, droplet based methods are frequently used. Single-cell transcriptomic technologies such as Drop-Seq (Macosko et al., Cell, 2015) and
In-Drop (Klein et al., Cell, 2015), have now also found their way to commercial platforms, which is where the Chromium platform from the company 10x Genomics is making waves
(Zheng et al., in press, 2016).

The Chromium instrument allows droplet-based loading of more than 48,000 single cells per experiment. Individual cells are collected in droplets containing barcoded oligos on beads, whereby up to a million different barcoded beads can be used per experiment, ensuring that each cell will have a unique matching
bead and oligo. Cells are lysed in the droplets and the mRNAs are captured on the beads, with cDNA synthesis performed in each droplet. After breaking the emulsion, a bulk library prep is made and the sample is ready for RNA-seq. After sequencing, the barcodes allow the sequencing reads to be grouped back to the mRNAs from individual cells.

Transcriptomic technology in the forecast
Even more exciting times are ahead for the transcriptome, since there are about a dozen companies that have plans to launch new instruments or platforms to look at gene expression. From direct RNA sequencing to detect novel base modifications to ultra-long reads for the comprehensive profiling of RNA-splicing, these
technologies will ensure exciting new discoveries.

Spatial Transcriptomics offers scientists novel insights into biology by enabling RNA sequencing in 2D