Menu

Close

SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem K. Saka, Yu Wang, Constance L. Cepko, Peng Yin

Preprint posted on August 27, 2018 https://www.biorxiv.org/content/early/2018/08/27/401810

Utilizing strand displacing polymerases, Kishi et al. forged SABER to enable a faster, more sensitive, and cheaper protocol of fluorescence in situ hybridization.

Selected by Yen-Chung Chen

Background and context

To understand where and how much a gene is expressed or where a genetic element locates on chromosomes, in situ hybridization is often the technique of choice. In situ hybridization involves detecting nucleic acid sequences with complementary probes labeled with isotopes, fluorophores, or enzymes [Reviewed in 1]. Though in situ hybridization is powerful and enables characterization and quantification of gene expression in tissues with single cell resolution, several challenges remain.

It is challenging to examine more than a couple of genes simultaneously due to the limitation of available fluorescent channels or chromogens, and signal intensity is an issue for in situ hybridization, where low intensity leads to indistinguishable signal in complicated tissues or an environment with auto-fluorescence.

To overcome these challenges, several modifications have been developed. A number of simultaneously detected targets can be increased using a combination of fluorophores on each of the probes [2, 3] or by labeling removal and re-labeling. Signal intensity can be amplified using a probe with “branched” binding site for tagging [4] or in situ amplification of detectable concatemers [5]. While these methods greatly broadened the application of in situ hybridization, the cost and complicated workflow can often limit the availability of these methods.

Key findings

Kishi et al. aimed to improve fluorescence in situ hybridization (FISH) by establishing a simple but robust protocol to synthesize probes tagged by repeated sequences (or concatemers) in a programmable fashion, so that multiple fluorophore-labeled “imager” oligonucleotides complementary to the concatemer can specifically bind to and light up the probe.

Utilizing primer exchange reaction, which they previously developed [6], the authors demonstrated that concatemers can be synthesized efficiently with defined lengths, and different concatemer sequences can be synthesized on the same probes by using different combination of catalytic DNA hairpin with primer exchange reaction. This programmability of concatemer length enables precise control of the degree and variability of signal amplification in FISH (Figure 1).

 

Figure 1. SABER-FISH: Amplifying FISH signal intensity with probes with programmable concatemer (Adapted from preprint figure 1C, available under a CC-BY-NC-ND 4.0 International license.)

 

The probes synthesized with this new technique, signal amplification by exchange reaction (SABER), were then tested on an MRC-5 cell model and retinal tissues. On MRC-5 cells, unbranched SABER provided 35 times stronger signal when detecting telomere DNA, compared to unextended FISH probes. When detecting Cbx5 mRNA, branched SABER provided even more powerful signal amplification: 464 times stronger than unextended FISH probes. On mouse retinal tissues, SABER was able to detect cell type markers specifically even with low expression level, and the signal intensity of each marker closely corresponded to relative abundance observed in single-cell RNA-seq.

In terms of the ability to multiplex, the authors showed that SABER is compatible with multiple rounds of labeling removal and relabeling and immunocytochemistry by simultaneously staining 7 transcripts with SABER along with two proteins with antibody. Furthermore, SABER is also compatible with using a combination of 3 fluorophores to perform 6-color labeling at the same time (Figure 2).

 

Figure 2. Multiplexing detection targets in SABER-FISH: Using multiple fluorescent images, re-labeling, and immunohistochemistry. (Adapted from preprint figure 5A, available under a CC-BY-NC-ND 4.0 International license.)

 

Finally, the authors examined potential enhancers for Grik1, a receptor enriched in OFF bipolar cells. Six enhancers were cloned into reporter constructs, driving expression of specific sequences detected with SABER. Two cell type-specific enhancers were identified, one driving expressing in Grik1-positive bipolar cells, and another driving expression in rod cells. Taking advantage of being able to detect DNA and RNA simultaneously, the authors also used SABER to quantify reporter transcripts and reporter plasmids at the same time to assess enhancer activity when accounting for plasmid number variation in each cell.

Why I like this preprint

The ease of manipulating nucleic acids makes in situ hybridization (ISH) time-efficient and powerful. Time from designing a new probe to experiment takes days instead of months. Nonetheless, the improvements to signal intensity and the ability to detect multiple targets require modifications that are not accessible to many labs, and thus the cost in time and resources to apply these improved ISH become similar to antibody-based detection methods.

In this preprint, Kishi et al. elegantly showed how strand displacing polymerase can be harnessed to simplify probe synthesis for signal-amplified FISH in a programmable and modular fashion. This technique allows precise control of concatemer length and a more defined degree of signal amplification. It is also compatible with fluorescence multiplexing and re-labeling, which enables potential high-throughput FISH on the same piece of tissue and the collection of expression profile with spatial resolution. FABER can potentially speed up and scale up research and clinical investigations relying on FISH and make the enhanced approach more accessible by lowering the cost of application.

Open question

  1. Several methods were developed to amplify FISH signal, and some of them used similar approach to enhance signal intensity per probe. I was wondering how well the signal intensity and specificity of SABER looks when compared to those.
  2. The authors showed that iterative branching concatemers enhance signal intensity by orders of magnitude in figure 2. This is a favorable feature when detecting lowly expressed transcripts, and since the probes used in figure 3 on are non-branching ones. I became curious about:
    1. Whether the signal from probes with iterative branching concatemers still closely reflects transcript abundance
    2. If probes with branching concatemers allow simultaneous detection of multiple probes

Reference

  1. Farrell, R. E. in RNA Methodologies 261–282 (Elsevier, 2010). doi:10.1016/B978-0-12-374727-3.00012-7
  2. Speicher, M. R., Ballard, S. G. & Ward, D. C. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12, 368 (1996).
  3. Schröck, E. et al. Multicolor Spectral Karyotyping of Human Chromosomes. Science (80-. ). 273, 494 LP-497 (1996).
  4. Player, A. N., Shen, L. P., Kenny, D., Antao, V. P. & Kolberg, J. A. Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J. Histochem. Cytochem. 49, 603–612 (2001).
  5. Dirks, R. M. & Pierce, N. A. Triggered amplification by hybridization chain reaction. Proc. Natl. Acad. Sci. U. S. A. 101, 15275 LP-15278 (2004).
  6. Kishi, J. Y., Schaus, T. E., Gopalkrishnan, N., Xuan, F. & Yin, P. Programmable autonomous synthesis of single-stranded DNA. Nat. Chem. 10, 155 (2017).

Tags: fish

Posted on: 19th September 2018 , updated on: 20th September 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the cell biology category:

    Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

    Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, et al.



    Selected by Lorenzo Lafranchi

    Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

    Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, et al.

    AND

    Live-cell single RNA imaging reveals bursts of translational frameshifting

    Kenneth R Lyon Jr, Luis U Aguilera, Tatsuya Morisaki, et al.



    Selected by Nicola Stevenson

    A non-canonical arm of UPRER mediates longevity through ER remodeling and lipophagy.

    Joseph R Daniele, Ryo Higuchi-Sanabria, Vidhya Ramachandran, et al.



    Selected by Sandra Malmgren Hill

    Microtubules Gate Tau Condensation to Spatially Regulate Microtubule Functions

    Ruensern Tan, Aileen J. Lam, Tracy Tan, et al.

    AND

    Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes

    Valerie Siahaan, Jochen Krattenmacher, Amayra Hernandez-Vega, et al.



    Selected by Satish Bodakuntla

    1

    The modular mechanism of chromocenter formation in Drosophila

    Madhav Jagannathan, Ryan Cummings, Yukiko M Yamashita



    Selected by Maiko Kitaoka

    1

    The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

    Joseph Franics McKenna, Daniel Rolfe, Stephen E D Webb, et al.



    Selected by Marc Somssich

    Psychiatric risk gene NT5C2 regulates protein translation in human neural progenitor cells

    Rodrigo R.R. Duarte, Nathaniel D. Bachtel, Marie-Caroline Cotel, et al.



    Selected by Joanna Cross

    Old fibroblasts secrete inflammatory cytokines that drive variability in reprogramming efficiency and may affect wound healing between old individuals

    Salah Mahmoudi, Elena Mancini, Alessandra Moore, et al.



    Selected by Shikha Nayar

    DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

    Joshua A. Weinstein, Aviv Regev, Feng Zhang



    Selected by Theo Sanderson

    2

    The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

    Federico Germani, Daniel Hain, Denise Sternlicht, et al.



    Selected by Rohan Khadilkar

    Drosophila kinesin-8 stabilises kinetochore-microtubule interaction

    Tomoya Edzuka, Gohta Goshima



    Selected by Ben Craske, Thibault Legal and Toni McHugh

    1

    Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

    Andrea Palamidessi, Chiara Malinverno, Emanuela FRITTOLI, et al.



    Selected by Tim Fessenden

    1

    Tension on kinetochore substrates is insufficient to prevent Aurora-triggered detachment

    Anna K de Regt, Charles L Asbury, Sue Biggins



    Selected by Angika Basant

    1

    Single-cell RNA sequencing reveals novel cell differentiation dynamics during human airway epithelium regeneration

    Sandra Ruiz Garcia, Marie Deprez, Kevin Lebrigand, et al.



    Selected by Rob Hynds

    1

    Actomyosin-driven tension at compartmental boundaries orients cell division independently of cell geometry in vivo

    Elena Scarpa, Cedric Finet, Guy Blanchard, et al.



    Selected by Ivana Viktorinová

    Molecular organization of integrin-based adhesion complexes in mouse Embryonic Stem Cells

    Shumin Xia, Evelyn K.F. Yim, Pakorn Kanchanawong

    AND

    Superresolution architecture of pluripotency guarding adhesions

    Aki Stubb, Camilo Guzmán, Elisa Närvä, et al.



    Selected by Nicola Stevenson, Amanda Haage

    Also in the neuroscience category:

    Distributed correlates of visually-guided behavior across the mouse brain

    Nicholas Steinmetz, Peter Zatka-Haas, Matteo Carandini, et al.



    Selected by Craig Bertram

    Psychiatric risk gene NT5C2 regulates protein translation in human neural progenitor cells

    Rodrigo R.R. Duarte, Nathaniel D. Bachtel, Marie-Caroline Cotel, et al.



    Selected by Joanna Cross

    Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord

    Julien Delile, Teresa Rayon, Manuela Melchionda, et al.



    Selected by Reena Lasrado

    1

    Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing

    Qingyun Li, Zuolin Cheng, Lu Zhou, et al.



    Selected by Zheng-Shan Chong

    SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

    Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, et al.



    Selected by Yen-Chung Chen

    LCM-seq reveals unique transcriptional adaption mechanisms of resistant neurons in spinal muscular atrophy

    Susanne Nichterwitz, Helena Storvall, Jik Nijssen, et al.

    AND

    Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS

    Jik Nijssen, Julio Cesar Aguila Benitez, Rein Hoogstraaten, et al.



    Selected by Yen-Chung Chen

    Simultaneous production of diverse neuronal subtypes during early corticogenesis

    Elia Magrinelli, Robin Jan Wagener, Denis Jabaudon



    Selected by Boyan Bonev

    1

    Local protein synthesis in axon terminals and dendritic spines differentiates plasticity contexts

    Anne-Sophie Hafner, Paul Donlin-Asp, Beulah Leitch, et al.



    Selected by Dipen Rajgor

    Moving beyond P values: Everyday data analysis with estimation plots

    Joses Ho, Tayfun Tumkaya, Sameer Aryal, et al.



    Selected by Gautam Dey

    1

    Optogenetic manipulation of medullary neurons in the locust optic lobe

    Hongxia Wang, Richard B. Dewell, Markus U. Ehrengruber, et al.



    Selected by Ana Patricia Ramos

    Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon

    Jessica Messier, Hongmei Chen, Zhao-Lin Cai, et al.

    AND

    High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins

    Mathias Mahn, Lihi Gibor, Katayun Cohen-Kashi Malina, et al.



    Selected by Mahesh Karnani

    2

    Phenotypic landscape of schizophrenia-associated genes defines candidates and their shared functions

    Summer B. Thyme, Lindsey M. Pieper, Eric H. Li, et al.



    Selected by Daniel Grimes

    Cerebellar contribution to preparatory activity in motor neocortex

    Francois Pierre Chabrol, Antonin Blot, Thomas D Mrsic-Flogel



    Selected by Mahesh Karnani

    1

    Fbxw7 is a critical regulator of Schwann cell myelinating potential

    Breanne L Harty, Fernanda Coelho, Sarah D Ackerman, et al.



    Selected by Yen-Chung Chen

    OptoGranules reveal the evolution of stress granules to ALS-FTD pathology

    Peipei Zhang, Baochang Fan, Peiguo Yang, et al.



    Selected by Srivats Venkataramanan

    1

    Mouse dLGN receives input from a diverse population of retinal ganglion cells with limited convergence

    Miroslav Román Rosón, Yannik Bauer, Philipp Berens, et al.



    Selected by Polona Jager
    Close