Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.
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.
Chen
Antony K
AK
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Behlke
Mark A
MA
Tsourkas
Andrew
A
D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D016477:Artifacts; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D045663:Quantum Dots; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D018931:Antineoplastic Agents, Hormonal; D005456:Fluorescent Dyes; C000715470:MAS1 protein, human; D015345:Oligonucleotide Probes; D011506:Proteins; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D013629:Tamoxifen; D004720:Endonucleases; D005092:Exonucleases
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