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Flow cytometry has long been the standard for characterizing heterogeneous cell populations, thanks to its ability to rapidly acquire and analyze millions of individual cells and to multiplex and detect both cell-surface and intracellular proteins in a straightforward workflow. One major limitation, however, is the availability of antibodies to measure all analytes of interest. Researchers studying microRNA (miRNA), long noncoding RNA (lncRNA), messenger RNA (mRNA), viral transcripts (vRNA), unique model organisms, or other targets for which antibody development is troublesome have not been able to utilize the power of flow cytometry. Historically they have had to conduct numerous independent experiments, often on bulk samples, to analyze these targets.
With the Invitrogen PrimeFlow RNA Assay Kit, researchers can now reveal the dynamics of RNA transcription together with protein expression patterns by flow cytometry. The PrimeFlow RNA assay employs fluorescence in situ hybridization (FISH) with branched DNA (bDNA) signal amplification for the simultaneous detection of up to four RNA targets, and it can be used in combination with immunolabeling of both cell-surface and intracellular proteins using fluorophore-conjugated antibodies.
In the PrimeFlow RNA assay workflow, cells are first labeled with cell-surface antibodies, fixed and permeabilized, and then labeled with intracellular antibodies. These cells are then sequentially hybridized with probes specific for the RNA targets, and hybridized targets are detected after bDNA signal amplification. In the initial hybridization step, a gene-specific oligonucleotide target probe set that contains 20–40 probe pairs (or a single pair in the case of miRNA probe sets) binds to the target RNA sequence. An individual probe pair contains two oligonucleotides that are designed to bind adjacent to each other on the RNA transcript for bDNA signal amplification to take place.
bDNA signal amplification is achieved through a series of sequential hybridization steps with preamplifiers, amplifiers, and then fluorophore-conjugated label probes. The preamplifier molecules confer an additional level of specificity because they will hybridize to the RNA target only after both members of the oligonucleotide target probe set have bound to their target sequence. Multiple amplifier molecules subsequently hybridize to their respective preamplifier molecules. Finally, label probe oligonucleotides, which are conjugated to a fluorescent dye, hybridize to their corresponding amplifier molecules. A fully assembled signal amplification “tree” has 400 label probe binding sites. When all target-specific oligonucleotides in the probe set bind to the target RNA transcript, 8,000- to 16,000-fold amplification can be achieved.
The PrimeFlow RNA assay currently offers four unique amplification structures that allow simultaneous measurement of up to four different RNA targets for multicolor flow cytometry analysis. Alexa Fluor 647 (Type 1) and Alexa Fluor 568 (Type 10) probe sets provide the most sensitive detection of the four sets and should be used for target genes with low or unknown levels of expression. Alexa Fluor 488 (Type 4) and Alexa Fluor 750 (Type 6) probe sets are designed for detecting target genes with medium to high levels of expression. The lower sensitivity of Alexa Fluor 488 probe sets, as compared with Alexa Fluor 647 probe sets, is in part due to potentially high levels of cell autofluorescence at the Alexa Fluor 488 detection wavelengths.
Once RNA targets have been hybridized and amplified using the PrimeFlow RNA Assay Kit, the cells can be analyzed on a standard flow cytometer. Figure 1 illustrates the workflow for the simultaneous detection of two unique RNA targets. With target-specific probe sets, the PrimeFlow RNA assay can be used to detect miRNA, lncRNA, and mRNA, as well as vRNA and telomere DNA.
Figure 1. The PrimeFlow RNA assay workflow. The workflow for the Invitrogen PrimeFlow RNA Assay Kit starts with optional antibody labeling, followed by fixation and permeabilization, and then hybridization with gene-specific target probes. This hybridization is then detected after branched DNA (bDNA) signal amplification using preamplifiers, amplifiers, and label probes. Labeled cells are analyzed on a standard flow cytometer.
Approximately 75% of the human genome can be transcribed into RNA; however, only 1.5% of the human genome codes for mRNA, which is typically translated into protein. Even though they do not encode proteins, nontranslated (or noncoding) RNA sequences often show tissue-specific expression and may contain sequences conserved across species, suggesting that they play a role in cell function. For example, miRNA sequences serve as key translational regulators for 30% of all protein-coding genes in diverse biological processes [1,2].
With advances in transcriptomic techniques, researchers have been able to identify, profile, validate, and functionally analyze relevant noncoding RNA in different models and diseases. However, analysis at the single-cell level, especially for miRNA, has been limited by low sensitivity and poor resolution. For detection of miRNA and other small RNA, we recommend the use of the Invitrogen PrimeFlow microRNA Pretreatment Buffer and the accompanying protocol. The use of this buffer with the PrimeFlow RNA assay improves the retention of some small RNA targets, resulting in better detection sensitivity. Figure 2 demonstrates that the PrimeFlow RNA Assay Kit, in combination with the PrimeFlow microRNA Pretreatment Buffer, enables the detection of miRNA together with antibody-based immunophenotyping.
Figure 2. PrimeFlow RNA assay detection of miR-146a, Arg1 mRNA, Cxcl13 mRNA, and Retnla mRNA in mouse peritoneal cells. C57BI/6 mouse resident peritoneal exudate cells were analyzed using the Invitrogen PrimeFlow RNA Assay Kit. Cells were stained with eFluor 450 anti–mouse F4/80 and PE-Cyanine7 anti–mouse CD11b antibodies, and fixed and permeabilized using PrimeFlow RNA Assay Kit buffers and protocols. Next, cells were hybridized with target-specific oligonucleotides using Type 1 human/mouse miR146a Alexa Fluor 647, Type 4 mouse Arg1 (arginase 1) Alexa Fluor 488, Type 6 mouse Cxcl13 Alexa Fluor 750, and Type 10 mouse Retn1a (Relm α) Alexa Fluor 568 target probes. Viable CD11b+ cells were used for analysis. The data show that both small peritoneal macrophages (SPM, F4/80–) and large peritoneal macrophages (LPM, F4/80+) were positive for miR-146a. SPM expressed high levels of Retnla (Relm α) mRNA, whereas LPM were positive for Cxcl13 mRNA and expressed low levels of Arg1 (arginase 1) mRNA.
The ability to measure RNA and protein expression together in individual cells provides a means of correlating their levels with cell function over time or in response to a stimulus. The PrimeFlow RNA Assay Kit provides a complete buffer system, compensation kit, and reagents for detecting up to 4 RNA transcripts in mammalian cells optionally labeled with antibodies that recognize cell-surface or intracellular proteins.
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