Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry

Abstract

Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image‐based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not possible to measure intracellular mRNA in specific phenotypes of cells by flow cytometry. In this protocol, a method of completing simultaneous intracellular measurement of mRNA and protein for PPAR‐gamma in peripheral blood monocytes, which have been exposed in vitro to modified LDL, is described. The process of PPAR‐gamma activation following uptake of modified LDL is believed to play a role in the development of atherogenesis. PPAR‐gamma mRNA measurement was made possible using an amplified FISH technique (PrimeFlow RNA Assay) that allowed for detection of low‐abundant intracellular mRNA expression. This protocol represents a continued effort by the authors’ laboratory to establish and validate new techniques to assess the role of the immune system in chronic disease. © 2016 by John Wiley & Sons, Inc.

Human lung tumor FOXP+ Tregs upregulate four “Treg-locking” transcription factors

“…Given an absence of specific membrane markers, human Tregs cannot be isolated for standard qPCR with high purity, an issue that is even more complicated for lung tissue samples ( Figure 5C). To resolve this problem, we applied the PrimeFlow method, which allows simultaneous measurement of mRNA and protein by flow cytometry (23) (Figure 8A and Supplemental Figure 41). By gating on FOXP3 + cells, we were able to evaluate mRNA expression of target genes in human Tregs that were 100% pure.…”

Cancer‐related mRNA expression analysis using a novel flow cytometry‐based assay

“…Notwithstanding the abovementioned results pave the way for routine diagnostic applications, the high labourintensity of this technique remains a hurdle (21). However, postponing FCM analysis for 72 h or even more, confirmed by our experimental data, makes it more user-friendly.…”

“…Expression of a NB-irrelevant target gene TARP, described in prostate and breast carcinoma (21), showed a highly comparable fluorescence intensity to the background fluorescence of the no-probe control (Supporting Information Fig. S2A versus S2B, respectively) in SK-N-BE(2)-C cells, that could clearly be distinguished from specific MYCN expression, hereby excluding off-target effects.…”

“…Only a handful of reports have documented its application and, to the best of our knowledge, cancer-related research has not yet been addressed (20)(21)(22)(23)(24)(25). Herein, the single-cell resolution of FCM is combined with a hybridization-based branched DNA (bDNA) technology, allowing intracellular RNA detection followed by exponential signal amplification.…”

“…Herein, the single-cell resolution of FCM is combined with a hybridization-based branched DNA (bDNA) technology, allowing intracellular RNA detection followed by exponential signal amplification. Only a handful of reports have documented its application and, to the best of our knowledge, cancer-related research has not yet been addressed (20)(21)(22)(23)(24)(25). Here, we evaluated the feasibility of PrimeFlow TM RNA assay in detecting key target mRNAs in NB and AML, using cell lines and patient samples, respectively.…”