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Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.

ABSTRACT: There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB-QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.

SUBMITTER: Chen AK

PROVIDER: S-EPMC2018645 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.

Chen Antony K AK Behlke Mark A MA Tsourkas Andrew A

Nucleic acids research 20070815 16

There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs ...[more]

PMID: 17702767

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Project description:Background: Single-cell RNA-seq is a powerful tool for measuring gene expression at the resolution of individual cells. A challenge in the analysis of this data is the large amount of zero values, representing either missing data or no expression. Several imputation approaches have been proposed to address this issue, but they generally rely on structure inherent to the dataset under consideration they may not provide any additional information, hence, are limited by the information contained therein and the validity of their assumptions. Methods: We evaluated the risk of generating false positive or irreproducible differential expression when imputing data with six different methods. We applied each method to a variety of simulated datasets as well as to permuted real single-cell RNA-seq datasets and consider the number of false positive gene-gene correlations and differentially expressed genes. Using matched 10X and Smart-seq2 data we examined whether cell-type specific markers were reproducible across datasets derived from the same tissue before and after imputation. Results: The extent of false-positives introduced by imputation varied considerably by method. Data smoothing based methods, MAGIC, knn-smooth and dca, generated many false-positives in both real and simulated data. Model-based imputation methods typically generated fewer false-positives but this varied greatly depending on the diversity of cell-types in the sample. All imputation methods decreased the reproducibility of cell-type specific markers, although this could be mitigated by selecting markers with large effect size and significance. Conclusions: Imputation of single-cell RNA-seq data introduces circularity that can generate false-positive results. Thus, statistical tests applied to imputed data should be treated with care. Additional filtering by effect size can reduce but not fully eliminate these effects. Of the methods we considered, SAVER was the least likely to generate false or irreproducible results, thus should be favoured over alternatives if imputation is necessary.

Project description:BackgroundMicroparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1?m. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.Materials and methodsWe analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).ResultsAnnexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).ConclusionWe observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

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Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.

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Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.

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