Isotachophoresis-based RNA extraction from fixed single cells

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

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An optimised direct lysis method for gene expression studies on low cell numbers

Scientific Reports ◽

10.1038/srep12859 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 6

Author(s):

Anh Viet-Phuong Le ◽

Dexing Huang ◽

Tony Blick ◽

Erik W. Thompson ◽

Alexander Dobrovic

Keyword(s):

Gene Expression ◽

Reverse Transcription ◽

Single Cells ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Cost Effective ◽

Breast Cancer Cell Lines ◽

Cell Numbers ◽

Ionic Detergent ◽

Gene Expression Studies

Abstract There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

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cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

10.17504/protocols.io.b3ifqkbn ◽

2022 ◽

Author(s):

Joost S S. Mansour ◽

Konstantinos Anestis ◽

Fabrice Not ◽

Uwe John

Keyword(s):

Single Cell ◽

Cdna Library ◽

Mammalian Cells ◽

Single Cells ◽

Rna Extraction ◽

Cdna Libraries ◽

Prymnesium Parvum ◽

Large Variability ◽

Total Rna ◽

Marine Protists

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

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A simplified method for isolation of large numbers of defined nephron segments

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.4.f650 ◽

1997 ◽

Vol 273 (4) ◽

pp. F650-F657 ◽

Cited By ~ 25

Author(s):

J. A. Schafer ◽

M. L. Watkins ◽

L. Li ◽

P. Herter ◽

S. Haxelmans ◽

...

Keyword(s):

Protein Extraction ◽

Single Cells ◽

Rna Extraction ◽

Nephron Segments ◽

Large Numbers ◽

Simplified Method ◽

Collecting Tubule ◽

Polymerase Chain ◽

Distinct Separation ◽

Normal Architecture

We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37°C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381–387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.

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Pairing Microwell Arrays with an Affordable, Semiautomated Single-Cell Aspirator for the Interrogation of Circulating Tumor Cell Heterogeneity

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630319898146 ◽

2020 ◽

Vol 25 (2) ◽

pp. 162-176

Author(s):

Jacob J. Tokar ◽

Charlotte N. Stahlfeld ◽

Jamie M. Sperger ◽

David J. Niles ◽

David J. Beebe ◽

...

Keyword(s):

Tumor Cell ◽

Single Cell ◽

Single Cells ◽

Rna Extraction ◽

Circulating Tumor Cell ◽

Bulk Sample ◽

Live Cells ◽

Target Cells ◽

Castration Resistant Prostate Cancer ◽

Background Population

Comprehensive analysis of tumor heterogeneity requires robust methods for the isolation and analysis of single cells from patient samples. An ideal approach would be fully compatible with downstream analytic methods, such as advanced genomic testing. These endpoints necessitate the use of live cells at high purity. A multitude of microfluidic circulating tumor cell (CTC) enrichment technologies exist, but many of those perform bulk sample enrichment and are not, on their own, capable of single-cell interrogation. To address this, we developed an affordable semiautomated single-cell aspirator (SASCA) to further enrich rare-cell populations from a specialized microwell array, per their phenotypic markers. Immobilization of cells within microwells, integrated with a real-time image processing software, facilitates the detection and precise isolation of targeted cells that have been optimally seeded into the microwells. Here, we demonstrate the platform capabilities through the aspiration of target cells from an impure background population, where we obtain purity levels of 90%–100% and demonstrate the enrichment of the target population with high-quality RNA extraction. A range of low cell numbers were aspirated using SASCA before undergoing whole transcriptome and genome analysis, exhibiting the ability to obtain endpoints from low-template inputs. Lastly, CTCs from patients with castration-resistant prostate cancer were isolated with this platform and the utility of this method was confirmed for rare-cell isolation. SASCA satisfies a need for an affordable option to isolate single cells or highly purified subpopulations of cells to probe complex mechanisms driving disease progression and resistance in patients with cancer.

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Mouse intestinal organoid time-course experiments from single cells

10.21203/rs.2.9220/v1 ◽

2019 ◽

Author(s):

Denise Serra ◽

Urs Mayr ◽

Andrea Boni ◽

Ilya Lukonin ◽

Markus Rempfler ◽

...

Keyword(s):

Stem Cells ◽

High Throughput ◽

Time Course ◽

Single Cells ◽

Rna Extraction ◽

Cell Types ◽

The Self ◽

Organoid Culture ◽

Tissue Organization ◽

Grown Structure

Abstract Organoids recapitulate the self-organizing capacity of stem cells and the tissue organization of the original organ in a controlled and trackable environment. Intestinal organoids, in particular, can develop from a single cell into a fully-grown structure that contains most of the cell types, patterns, and morphogenetic properties of the adult intestine. Here we present a protocol for high-throughput organoid culture in multi-well plate format combined with high content immunofluorescence imaging and RNA extraction. Our protocol allows recording and analysis of thousands of organoids during several days of development.

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Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

10.1101/2020.10.05.326082 ◽

2020 ◽

Author(s):

Prasanna Channathodiyil ◽

Jonathan Houseley

Keyword(s):

Flow Cytometry ◽

Transcriptome Analysis ◽

Mammalian Cells ◽

Single Cells ◽

Rna Extraction ◽

Cyclin B1 ◽

Immunofluorescent Staining ◽

Rna Seq ◽

Simple Method ◽

Staining Methods

AbstractA simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal - a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

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Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate)

Analytical Chemistry ◽

10.1021/acs.analchem.8b02338 ◽

2018 ◽

Vol 90 (21) ◽

pp. 12512-12518 ◽

Cited By ~ 6

Author(s):

Sangamithirai Subramanian Parimalam ◽

Yusuke Oguchi ◽

Mahmoud N. Abdelmoez ◽

Arata Tsuchida ◽

Yuka Ozaki ◽

...

Keyword(s):

Single Cells ◽

Rna Extraction ◽

Electrical Lysis

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Single cell high-throughput qRT-PCR protocol

10.21203/rs.3.pex-919/v1 ◽

2021 ◽

Author(s):

Sirisha Achanta ◽

Rajanikanth Vadigepalli

Keyword(s):

Single Cell ◽

High Throughput ◽

Reverse Transcription ◽

Single Cells ◽

Rna Extraction ◽

High Sensitivity ◽

Single Experiment ◽

Tissue Samples ◽

Qrt Pcr ◽

Cell Sample

Abstract Single cell high-throughput qRT-PCR protocol combines high sensitivity technique of single cell qPCR with high-throughput qPCR technology that can generate data from 96 samples and 96 genes in a single experiment. It can be adapted for various sample types- cell culture, tissue samples and extracted RNA (10 pg) and measured on traditional qPCR and high-throughput qPCR platforms. The workflow is comprised of four steps – cell lysis, reverse transcription, pre-amplification and qPCR. Key features of this protocol are; processing low input samples directly to reverse transcription without RNA extraction which minimizes sample loss, pre-amplification enables amplification of cDNA from single cells to detectable levels for qPCR and measuring up to 400 genes from a single cell sample/10 pg of RNA (starting material). Robust, reproducible and versatile this protocol can be adapted to several upstream and downstream techniques.

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Effects of carbamylcholine on chick embryo aggregate retina cell cultures

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103814 ◽

1988 ◽

Vol 46 ◽

pp. 350-351

Author(s):

Glenn M. Cohen ◽

Radharaman Ray

Keyword(s):

Cell Cultures ◽

Single Cells ◽

Retinal Cell ◽

Cell Aggregates ◽

High Concentration ◽

In Ovo ◽

Sterile Culture ◽

Retinal Tissue ◽

Synaptic Junctions ◽

And Control

Retinal,cell aggregates develop in culture in a pattern similar to the in ovo retina, forming neurites first and then synapses. In the present study, we continuously exposed chick retinal cell aggregates to a high concentration (1 mM) of carbamylcholine (carbachol), an acetylcholine (ACh) analog that resists hydrolysis by acetylcholinesterase (AChE). This situation is similar to organophosphorus anticholinesterase poisoning in which the ACh level is elevated at synaptic junctions due to inhibition of AChE, Our objective was to determine whether continuous carbachol exposure either damaged cholino- ceptive neurites, cell bodies, and synaptic elements of the aggregates or influenced (hastened or retarded) their development.The retinal tissue was isolated aseptically from 11 day embryonic White Leghorn chicks and then enzymatically (trypsin) and mechanically (trituration) dissociated into single cells. After washing the cells by repeated suspension and low (about 200 x G) centrifugation twice, aggregate cell cultures (about l0 cells/culture) were initiated in 1.5 ml medium (BME, GIBCO) in 35 mm sterile culture dishes and maintained as experimental (containing 10-3 M carbachol) and control specimens.

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