Application of an RNA Amplification Method For Reliable Single-Cell Transcriptome Analysis

Suslov, Oleg; Silver, Daniel J.; Siebzehnrubl, Florian A.; Orjalo, Arturo V.; Ptitsyn, Andrey A.; Steindler, Dennis A.

doi:10.2144/000114331

Cited by 4 publications

(4 citation statements)

References 49 publications

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“…Recently, a novel method was developed that combines exponential amplification of a limited number of PCR cycles with linear amplification of T7-based IVT for RNA amplification (38). The novelty of this technique lies in that it allows for high-fidelity gene expression profiling of individual cells and for significant reduction of 3'-bias; this enables detection of all regions of RNA transcripts.…”

Section: Single-cell Rna Amplification and Sequencing Methodsmentioning

confidence: 99%

“…To achieve this objective, some protocol modifications have been made. These modifications include: (1) the introduction of “extending primers” that contain a Kozak sequence at the 3′-ends during PCR which allows capture of 5′-ends of a gene's coding sequence; (2) the combination of modified polyT and modified random primers to make the PCR step more efficient and to improve the preservation of relative gene abundance, which secures full-length RNA coverage, and eliminating carryover of reverse transcriptase that greatly diminishes reverse transcriptase inhibitory effect on the following PCR steps; (3) using a SmartSpec 3000 spectrophotometer (Bio-Rad), the RNA yield was evaluated, and the yield of amplified RNA came up to 200–250 μg from a single cell, which is sufficient to apply to any RNA sequencing technique (38). In a word, this new technique has great potential for future applications.…”

Section: Single-cell Rna Amplification and Sequencing Methodsmentioning

confidence: 99%

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Technical Advances in Single-Cell RNA Sequencing and Applications in Normal and Malignant Hematopoiesis

Huang

Qin

et al. 2018

Front. Oncol.

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Single-cell RNA sequencing (scRNA-seq) has been tremendously developed in the past decade owing to overcoming challenges associated with isolation of massive quantities of single cells. Previously, cell heterogeneity and low quantities of available biological material posed significant difficulties to scRNA-seq. Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of normal and malignant hematopoietic cells; this heterogeneity has often been ignored in omics studies. The application of scRNA-seq has profoundly changed our comprehension of many biological phenomena, including organ development and carcinogenesis. Hematopoiesis, is actually a maturation process for more than ten distinct blood and immune cells, and is thought to be critically involved in hematological homeostasis and in sustaining the physiological functions. However, aberrant hematopoiesis directly leads to hematological malignancy, and a deeper understanding of malignant hematopoiesis will provide deeper insights into diagnosis and prognosis for patients with hematological malignancies. Here, we aim to review the recent technical progress and future prospects for scRNA-seq, as applied in physiological and malignant hematopoiesis, in efforts to further understand the hematopoietic hierarchy and to illuminate personalized therapy and precision medicine approaches used in the clinical treatment of hematological malignancies.

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Section: Single-cell Rna Amplification and Sequencing Methodsmentioning

confidence: 99%

Section: Single-cell Rna Amplification and Sequencing Methodsmentioning

confidence: 99%

Technical Advances in Single-Cell RNA Sequencing and Applications in Normal and Malignant Hematopoiesis

Huang

Qin

et al. 2018

Front. Oncol.

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“…In the rodent brain, for example, presence of a functioning EGFR is thought to be necessary for the interaction of neural stem cells with their environment to maintain their self-renewal capabilities [ 16 ]. There is little direct evidence in humans for this [ 17 ], but in a rare investigation of human tissues over a wide age range, it appeared as if EGF and EGFR are relevant for a microglial sustained persistence or proliferation of subventricular zone neuroglial stem cells [ 18 ]. Circumstantial support may come from the limited experience using EGFR TKIs in addition to radiation for brain metastases, which resulted in increased survival but was associated with increased neurocognitive decline [ 19 ], possibly an effect on stem-cell-based regenerative capacities.…”

Section: Compartmental Selectivity Of Egfr Targetingmentioning

confidence: 99%

EGFR as a Target for Glioblastoma Treatment: An Unfulfilled Promise

2017

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The receptor for epidermal growth factor (EGFR) is a prime target for cancer therapy across a broad variety of tumor types. As it is a tyrosine kinase, small molecule tyrosine kinase inhibitors (TKIs) targeting signal transduction, as well as monoclonal antibodies against the EGFR, have been investigated as anti-tumor agents. However, despite the long-known enigmatic EGFR gene amplification and protein overexpression in glioblastoma, the most aggressive intrinsic human brain tumor, the potential of EGFR as a target for this tumor type has been unfulfilled. This review analyses the attempts to use TKIs and monoclonal antibodies against glioblastoma, with special consideration given to immunological approaches, the use of EGFR as a docking molecule for conjugates with toxins, T-cells, oncolytic viruses, exosomes and nanoparticles. Drug delivery issues associated with therapies for intracerebral diseases, with specific emphasis on convection enhanced delivery, are also discussed.

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“…To test the sensitivity of the Landscape + Breast Cancer assay, the gene expression levels using different concentrations (20, 40, 60, 80, and 100 pg) of total RNA from four different breast cancer cell lines (BT474-M1, MDA-MB-231, SK-BR-3 and MCF-7) and one ovarian cancer cell line RNA (Caov3) were measured in triplicate at each RNA concentration. The 20 pg RNA level represents the approximate amount of total RNA contained in a single cell [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer

et al. 2023

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Background Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system. Methods The HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs). Results Using genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs. Conclusion HyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.

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Application of an RNA Amplification Method For Reliable Single-Cell Transcriptome Analysis

Cited by 4 publications

References 49 publications

Technical Advances in Single-Cell RNA Sequencing and Applications in Normal and Malignant Hematopoiesis

Technical Advances in Single-Cell RNA Sequencing and Applications in Normal and Malignant Hematopoiesis

EGFR as a Target for Glioblastoma Treatment: An Unfulfilled Promise

Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer

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