Abstract
SmartFlare probes have recently emerged as a promising tool for visualisation and quantification of specific RNAs in living cells. They are supposed to overcome the common drawbacks of current methods for RNA analysis: the need of cell fixation or lysis, or the requirements for genetic manipulations. In contrast to the traditional methods, SmartFlare probes are also presumed to provide information on RNA levels in single cells. Disappointingly, the results of our comprehensive study involving probes specific to five different transcripts, HMOX1, IL6, PTGS2, Nrg1, and ERBB4, deny the usefulness of SmartFlare probes for RNA analysis. We report a total lack of correlation between fluorescence intensities of SmartFlare probes and the levels of corresponding RNAs assessed by RT-qPCR. To ensure strong differences in the levels of analysed RNAs, their expression was modified via: (i) HMOX1-knockdown generated by CRISPR-Cas9 genome editing, (ii) hemin-mediated stimulation of HMOX1- and IL1β-mediated stimulation of IL6- and PTGS2 transcription, (iii) lentiviral vector-mediated Nrg1 overexpression. Additionally, ERBB4-specific SmartFlare probe failed to distinguish between ERBB4-expressing and non-expressing cell lines. Finally, we demonstrated that fluorescence intensity of HMOX1-specific SmartFlare probe corresponds to the efficacy of its uptake and/or accumulation.
Highlights
The majority of quantitative analyses of expression of specific RNAs require cell fixation or lysis to isolate RNA; the cells are lost for further experiments
SmartFlare probes do not discriminate between cells that strongly differ in specific transcript levels
Deficient as a result of CRISPR-Cas9-mediated genome editing. 293T cells were transiently transfected with HMOX1-targeting pX330-puromycin N-acetyltransferase (Pac)-Cerulean fluorescent protein (Cer) vector coding for human codon-optimized Cas[9] and human HMOX1-specific sgRNA or with control empty pX330-Pac-Cer
Summary
The majority of quantitative analyses of expression of specific RNAs require cell fixation or lysis to isolate RNA; the cells are lost for further experiments. A method that enables detection of a specific RNA in single living cells would be highly desirable. A novel tool for RNA detection in living cells, namely SmartFlare, has been brought onto the market. It is based on previously developed Nanoflare technology[3]. SmartFlare probes are ~13 nm gold nanoparticle-coupled single-stranded DNA (ssDNA) oligonucleotides designed to selectively bind a desired transcript. We present a comprehensive evaluation of SmartFlare usefulness to analyse specific transcript levels in living cells. We compared SmartFlare HMOX1-Cy5, IL6-Cy5, PTGS2-Cy5, and Nrg1-Cy5 fluorescence intensities with the levels of corresponding mRNAs evaluated by RT-qPCR. We show that HMOX1-Cy5, IL6-Cy5, PTGS2-Cy5 and Nrg1-Cy5 SmartFlare fluorescence intensities do not correlate with mRNA levels measured by RT-qPCR. We provide evidence that within the same cell type, SmartFlare fluorescence intensity is associated with the ability of the cells to internalize/accumulate SmartFlare probes
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