SmartFlares fail to reflect their target transcripts levels

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.

Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity

“…TWIST1 transcripts were analysis by RT-PCR. Values represent the mean ± SD from duplicates or quadruplicate uptake capacity can influence the SmartFlare signal specificity (Czarnek and Bereta 2017), we decided to carefully monitor uptake in our BMSC populations.…”

“…Multiple studies successfully detected mRNA expression with the SmartFlare technique (McClellan et al 2015;Kronig et al 2015;Lahm et al 2015;Seftor et al 2014;Li et al 2016). However, two recent studies showed that different SmartFlare probes were not able to specifically detect their target mRNAs in cell lines and monocytes (Czarnek and Bereta 2017;Yang et al 2018). Here we showed that SmartFlare is an effective tool to detect TWIST1 gene expression in living BMSCs, but differences in probe concentration, incubation time and cellular uptake can influence the SmartFlare sensitivity and possibly lead to misinterpretation of the results.…”

“…However, other studies did not find a correlation between the SmartFlare fluorescence intensity and mRNA expression measured by RT-PCR (Czarnek and Bereta 2017;Yang et al 2018). In addition, Czarnek et al (2017) found that the SmartFlare signal intensity correlates with the probe uptake ability of the cells.…”

“…Additionally it was applied to investigate a Nodal expressing subpopulation of melanoma cells (Seftor et al 2014), and to study a subpopulation of human BMSCs with an enhanced osteogenic potential (Li et al 2016). However, other studies did not find a correlation between the SmartFlare fluorescence intensity and mRNA expression measured by RT-PCR (Czarnek and Bereta 2017;Yang et al 2018). In addition, Czarnek et al (2017) found that the SmartFlare signal intensity correlates with the probe uptake ability of the cells.…”

Enrichment of Skeletal Stem Cells from Human Bone Marrow Using Spherical Nucleic Acids

“…We used the PODXL nanoprobe as the uptake control in the current studies, to determine the one-hour contact time as this used the same chemistry as all other nanoprobes examined. The common uptake control is a directly labelled, extended length sense strand as this fluoresces at all times 21 . In the current studies, we observed negligible FAM fluorescence suggesting minimal DNA degradation of the nanoprobes.…”