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Related Concept Videos

Targeted Cancer Therapies02:57

Targeted Cancer Therapies

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The targeted cancer therapies, also known as “molecular targeted therapies,” take advantage of the molecular and genetic differences between the cancer cells and the normal cells. It needs a thorough understanding of the cancer cells to develop drugs that can target specific molecular aspects that drive the growth, progression, and spread of cancer cells without affecting the growth and survival of other normal cells in the body.
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Cancers Originate from Somatic Mutations in a Single Cell02:21

Cancers Originate from Somatic Mutations in a Single Cell

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Cancer arises from mutations in the critical genes that allow healthy cells to escape cell cycle regulation and acquire the ability to proliferate indefinitely. Though originating from a single mutation event in one of the originator cells, cancer progresses when the mutant cell lines continue to gain more and more mutations, and finally, become malignant. For example, chronic myelogenous leukemia (CML) develops initially as a non-lethal increase in white blood cells, which progressively...
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Transcription01:10

Transcription

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Overview
Transcription is the process of synthesizing RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. Additionally, many other proteins and regulatory sequences are involved in the proper synthesis of messenger RNA (mRNA). Regulation of transcription is responsible for the differentiation of all the different types of cells and often for the proper cellular response to environmental signals.
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Transcription Factors02:16

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Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
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Eukaryotic Transcription Activators02:42

Eukaryotic Transcription Activators

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Transcription activators are proteins that promote the transcription of genes from DNA to RNA. In most cases, these proteins contain two separate domains ‒ a domain that binds to DNA and a domain for activating transcription; however, in some cases, a single domain is responsible for both binding and activation of transcription, as seen in the glucocorticoid receptor and MyoD.
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Master Transcription Regulators02:23

Master Transcription Regulators

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Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a  complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
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Related Experiment Video

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Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization
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Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

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Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification.

Franziska C Durst1, Ana Grujovic1, Iris Ganser1

  • 1Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany.

Plos One
|August 21, 2019
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Summary

We developed a sensitive quantitative PCR (qPCR) assay for analyzing single-cell gene expression. This method accurately measures ERBB2 gene expression in disseminated cancer cells (DCCs), aiding targeted therapy decisions.

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Area of Science:

  • Molecular Biology
  • Cancer Research
  • Genomics

Background:

  • Analyzing gene expression in rare or heterogeneous cell populations like disseminated cancer cells (DCCs) requires highly sensitive methods.
  • Existing techniques may lack the precision needed for reliable single-cell analysis, hindering the understanding of cancer cell heterogeneity and treatment response.

Purpose of the Study:

  • To develop and validate a quantitative PCR (qPCR) assay for accurate single-cell gene expression analysis.
  • To optimize a workflow for measuring ERBB2 gene expression in DCCs, particularly in the context of HER2-targeted therapies.

Main Methods:

  • Developed a single-cell quantitative PCR (qPCR) assay utilizing pre-amplified cDNA from whole transcriptome amplification (WTA).
  • Optimized key steps including WTA product re-amplification, cDNA yield quantification, and final qPCR analysis.
  • Employed absolute quantification over relative quantification for enhanced accuracy in ERBB2 gene expression measurement.

Main Results:

  • The optimized single-cell qPCR assay demonstrated high reliability and accuracy in quantifying ERBB2 gene expression.
  • Validation on breast cancer cell lines showed that transcript levels accurately reflected distinct HER2 protein expression.
  • Successfully applied the method to measure ERBB2 expression in disseminated cancer cells from a patient undergoing anti-HER2 therapy.

Conclusions:

  • A robust and accurate single-cell qPCR assay was established for quantifying gene expression in DCCs.
  • The developed method enables precise measurement of ERBB2 levels in individual cancer cells, supporting personalized anti-HER2 treatment strategies.
  • This approach provides a valuable tool for studying cancer cell heterogeneity and monitoring treatment efficacy at the single-cell level.