Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR)

Brail, Les; Jang, Anne; Billia, Filio; Iscove, Norman N.; Klamut, Henry J.; Hill, Richard P.

doi:10.1016/s1383-5726(98)00009-0

Cited by 34 publications

(25 citation statements)

References 21 publications

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“…However, an RNA amplification procedure is requisite to generate significant hybridization signal intensity for cDNA microarray platforms. PCR is not suitable for this application because exponential amplification cannot preserve optimally the quantitative relationships between the expressed genes, 5,25 a parameter critical for expression profiling. Hybrid protocols using PCR-based technology combined with aRNA have yielded positive results.…”

Section: Resultsmentioning

confidence: 99%

Amplification of RNA transcripts using terminal continuation

Che

Ginsberg

2003

Lab Invest

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A new methodology has been developed to amplify RNA from minute amounts of starting material. Specifically, an efficient means of second-strand (ss) cDNA synthesis using a sequence-specific 'terminal continuation' (TC) method is demonstrated. An RNA synthesis promoter is attached to the 3 0 and/or 5 0 region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications including gene expression profiling, cDNA microarray analysis, and cDNA library/subtraction library construction. Synthesized sense TC-amplified RNA can also be used as a template for in vitro protein translations and downstream proteomic applications. The TC RNA amplification methodology offers high sensitivity, flexibility, and throughput capabilities. A likely mechanism is that the TC primer binds preferentially to GC-rich CpG islands flanking 5 0 regions of DNA that contain promoter sequences. Following TC RNA amplification, a large proportion of genes can be assessed quantitatively as evidenced by bioanalysis and cDNA microarray analysis in mouse and human postmortem brain tissues.

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Section: Resultsmentioning

confidence: 99%

Amplification of RNA transcripts using terminal continuation

Che

Ginsberg

2003

Lab Invest

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“…7 Amplification can also be a source of variability in the experimental procedure. 8 All three probe labeling approaches we used here involve an RT step, and all three probes had similar levels of discrepant gene detection. This argues that the discrepant values are due to sample complexity and RT, rather than amplification.…”

Section: Discussionmentioning

confidence: 99%

Reproducibility of Alternative Probe Synthesis Approaches for Gene Expression Profiling with Arrays

Vernon

Unger

Rajeevan

et al. 2000

The Journal of Molecular Diagnostics

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Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 g of purified poly(A)؉ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 g total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 g total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard. (J Mol Diag 2000, 2:124 -127)

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“…Standard RT-PCR can amplify genetic signals from small tissue samples. However, PCR-based methods tend to amplify abundant genes over rare genes and may distort quantitative relationships among gene populations due to exponential, nonlinear amplification (8,47). Realtime qRT-PCR lessens concerns regarding linear amplification and obviates the need for gel electrophoresis and the use of radioactivity for PCR quantification.…”

Section: Qpcr Using Small Sample Input Sourcesmentioning

confidence: 99%

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

et al. 2004

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Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by singlecell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively. KEY WORDS:Alzheimer's disease; cDNA microarray; cholinergic basal forebrain; dopamine receptors; expression profiling; protein phosphatases; RNA amplification; schizophrenia; single-cell microaspiration. Abbreviations (note the use of the NCBI-Unigene annotation): 3Rtau, three-repeat tau; 4Rtau, four-repeat tau; ACT, alpha-1-antichymotrypsin; ACTB, beta-actin; AGER, advanced glycosylation end product-specific receptor; APOE, apolipoprotein E; APP, amyloid-beta precursor protein; arc, activity regulated cytoskeletal-associated protein; BAX,

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Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR)

Cited by 34 publications

References 21 publications

Amplification of RNA transcripts using terminal continuation

Amplification of RNA transcripts using terminal continuation

Reproducibility of Alternative Probe Synthesis Approaches for Gene Expression Profiling with Arrays

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

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