Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

Lee, Je Hyuk; Daugharthy, Evan R; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas; Terry, Richard; Turczyk, Brian M.; Yang, Jason; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M.

doi:10.1038/nprot.2014.191

Cited by 455 publications

(374 citation statements)

References 52 publications

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“…The location of the affected astrocyte subpopulation can therefore already indicate the embryonically pattered subtype. New RNA sequencing methods, such as single cell sequencing (Marques et al, 2016) and fluorescence in situ sequencing of RNA (FISSEQ; (Lee et al, 2015)), will soon provide more insight into the glial subtypes and hopefully insight into the basis of selective vulnerability in VWM patients.…”

Section: Discussionmentioning

confidence: 99%

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

Leferink

Breeuwsma

Bugiani

et al. 2017

Glia

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Leukodystrophies are often devastating diseases, presented with progressive clinical signs as spasticity, ataxia and cognitive decline, and lack proper treatment options. New therapy strategies for leukodystrophies mostly focus on oligodendrocyte replacement to rescue lack of myelin in the brain, even though disease pathology also often involves other glial cells and the spinal cord. In this study we investigated spinal cord pathology in a mouse model for Vanishing White Matter disease (VWM) and show that astrocytes in the white matter are severely affected. Astrocyte pathology starts postnatally in the sensory tracts, followed by changes in the astrocytic populations in the motor tracts. Studies in post‐mortem tissue of two VWM patients, a 13‐year‐old boy and a 6‐year‐old girl, confirmed astrocyte abnormalities in the spinal cord. For proper development of new treatment options for VWM and, possibly, other leukodystrophies, future studies should investigate spinal cord involvement.

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Section: Discussionmentioning

confidence: 99%

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

Leferink

Breeuwsma

Bugiani

et al. 2017

Glia

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“…The efficiency for detecting single transcript molecules has been estimated to be 30% [13,16] and 3% [15] respectively, which is much lower than the 85% of hybridization efficiency in sm-FISH [4,17]. Such low efficiencies currently prevent these alternative methods from surveying the transcriptome with singlemolecule sensitivity and resolution in situ [18,19].…”

Section: Alternative Methods For Rna Detection In Imagingmentioning

confidence: 99%

“…Alternatively, transcripts can be visualized indirectly via reverse transcription to cDNA that can be sequenced in situ by padlock probes [13] or oligonucleotide ligation and detection [14,15].…”

Section: Alternative Methods For Rna Detection In Imagingmentioning

confidence: 99%

“…While the former sequencing approach can presently detect 31 different genes simultaneously in thousands of single cells within a tissue slide [13], the latter approach can currently read around 200 mRNAs simultaneously for 40 different cells [15]. The efficiency for detecting single transcript molecules has been estimated to be 30% [13,16] and 3% [15] respectively, which is much lower than the 85% of hybridization efficiency in sm-FISH [4,17].…”

Section: Alternative Methods For Rna Detection In Imagingmentioning

confidence: 99%

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Computer vision for image-based transcriptomics

Stoeger

Battich

Herrmann

et al. 2015

Methods

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Single-cell transcriptomics has recently emerged as one of the most promising tools for understanding the diversity of the transcriptome among single cells. Image-based transcriptomics is unique compared to other methods as it does not require conversion of RNA to cDNA prior to signal amplification and transcript quantification. Thus, its efficiency in transcript detection is unmatched by other methods. In addition, image-based transcriptomics allows the study of the spatial organization of the transcriptome in single cells at single-molecule, and, when combined with superresolution microscopy, nanometer resolution. However, in order to unlock the full power of image-based transcriptomics, robust computer vision of single molecules and cells is required. Here, we shortly discuss the setup of the experimental pipeline for image-based transcriptomics, and then describe in detail the algorithms that we developed to extract, at high-throughput, robust multivariate feature sets of transcript molecule abundance, localization and patterning in tens of thousands of single cells across the transcriptome. These computer vision algorithms and pipelines can be downloaded from: https://github.com/pelkmanslab/ImageBasedTranscriptomics. AbstractSingle-cell transcriptomics has recently emerged as one of the most promising tools for

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“…On the other hand, in situ hybridization allows simultaneous visualization of cellular location of a small number of transcripts 124.…”

Section: Next‐generation Sequencing‐based Methods For Global Investigmentioning

confidence: 99%

State of the art technologies to explore long non‐coding RNAs in cancer

Salehi

Taheri

Azarpira

et al. 2017

J Cellular Molecular Medi

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Long non‐coding RNAs (lncRNAs) comprise a vast repertoire of RNAs playing a wide variety of crucial roles in tissue physiology in a cell‐specific manner. Despite being engaged in myriads of regulatory mechanisms, many lncRNAs have still remained to be assigned any functions. A constellation of experimental techniques including single‐molecule RNA in situ hybridization (sm‐RNA FISH), cross‐linking and immunoprecipitation (CLIP), RNA interference (RNAi), Clustered regularly interspaced short palindromic repeats (CRISPR) and so forth has been employed to shed light on lncRNA cellular localization, structure, interaction networks and functions. Here, we review these and other experimental approaches in common use for identification and characterization of lncRNAs, particularly those involved in different types of cancer, with focus on merits and demerits of each technique.

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Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

Cited by 455 publications

References 52 publications

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

Computer vision for image-based transcriptomics

State of the art technologies to explore long non‐coding RNAs in cancer

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