基于 PrimeFlow<sup>TM</sup> 的临床前小鼠脾脏样本转基因 RNA 与蛋白细胞类型特异性同步检测

Sachith Gallolu Kankanamalage; Xiaoyu Fan; Sharmistha Kundu; Olga Villamizar; Jang Suk Roh; Justin Cohen; Hendrik Neubert; Polina Goihberg

doi:10.21769/BioProtoc.5748

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Peer-reviewed

PrimeFlow^TM Assay for Cell Type–Specific Co-detection of Transgene RNA and Protein in Mouse Spleens From Preclinical Studies

基于 PrimeFlow^TM 的临床前小鼠脾脏样本转基因 RNA 与蛋白细胞类型特异性同步检测

SK Sachith Gallolu Kankanamalage email

PG Polina Goihberg email

发布: 2026年06月20日第16卷第12期 DOI: 10.21769/BioProtoc.5748 浏览次数: 303

评审: Kif Liakath-AliSalma MerchantAnonymous reviewer(s)

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Abstract

The PrimeFlow^TM assay is a flow cytometry–based method for the co-detection of RNAs and proteins in cells. When combined with cell characterization by immunophenotyping, PrimeFlow^TM can be used to simultaneously detect RNA and proteins in a cell type–specific manner in complex heterogeneous samples, offering an advantage over bulk tissue analysis methods. Here, we describe the implementation of the PrimeFlow^TM assay protocol for the detection of transgene mRNA and protein expression in spleen samples from mice treated in vivo with luciferase mRNA-lipid nanoparticles (LNPs). This protocol involves spleen tissue dissociation for cell isolation, followed by cell fixation and permeabilization to allow immunolabeling of intracellular luciferase protein. The immunophenotyping strategy is based on immunolabeling with mouse CD marker antibodies for the identification of T cells, B cells, monocytes, granulocytes/macrophages, NK cells, and non-hematopoietic cells. The RNAs of luciferase and a housekeeping gene, β-actin, are detected with sequence-specific probe sets by employing sequential oligonucleotide annealing steps and fluorescent labeling using a branched DNA (bDNA) technology. Samples are analyzed by flow cytometry. Based on our analysis, we conclude it is feasible to apply the PrimeFlow^TM approach for evaluating successful drug targeting to the cell types of interest and any potential differences in the kinetics of RNA delivery and protein expression in various tissue cells, supporting the discovery and development of RNA therapeutics.

Key features

• This protocol can be employed for cell-specific simultaneous detection of up to four different RNAs (using available commercial reagents) and multiple proteins by flow cytometry.

• This protocol requires application-specific optimization of the RNA-binding probe sets and antibody reagents for transgene detection and immunophenotyping panel.

Keywords: PrimeFlow^TMassay (PrimeFlow^TM 检测)

Flow cytometry (流式细胞术)

RNA therapeutics (RNA 疗法)

Transgene expression (转基因表达)

Immunophenotyping (免疫表型分析)

RNA analysis (RNA 分析)

Flow-based in situ hybridization (基于流式的原位杂交)

Branched DNA (bDNA) (分支 DNA（bDNA）)

Graphical overview

Overview of the PrimeFlow^TM assay workflow for mouse spleen analysis. Cells are dissociated from the spleens of mRNA-lipid nanoparticle (LNP)-injected mice. Then, immunophenotyping with target protein labeling is conducted, followed by target-specific oligonucleotide probe hybridization and RNA signal amplification by bDNA reaction [1]. The cells are analyzed with flow cytometry. Created in Biorender.com.

Background

Development of gene therapies and vaccines relies on efficient transgene RNA delivery to target cell types, which necessitates a detailed understanding of RNA level and transgene protein expression kinetics in different cell populations. However, current bioanalytical methods, such as PCR for RNA and liquid chromatography–mass spectrometry (LC-MS) for protein measurements, usually do not allow simultaneous RNA and protein measurement in the same sample. More importantly, these methods rely on bulk tissue analysis and, therefore, do not provide cell type–specific resolution.

The PrimeFlow^TM technique, commercially available as Thermo Fisher Scientific PrimeFlow^TM RNA Assay, can potentially mitigate the above-mentioned issues. The PrimeFlow^TM assay has been developed by combining flow cytometry with in-situ hybridization and the branched DNA (bDNA) amplification technique [1–5]. The detection of target proteins, either membrane or intracellular, is conducted with fluorescently labeled antibodies that are found compatible with the assay conditions. The RNA detection by the PrimeFlow^TM approach requires in-situ hybridization with target-specific probe pairs, provided by Thermo Fisher, either off the shelf or custom-generated. Then, the bound probe signal is amplified (reportedly, ~8,000–16,000 fold [1–5]), through a series of steps: the bDNA reaction causes oligonucleotide pre-amplifiers to anneal the pre-bound target probe pairs, then multiple amplifiers bind each pre-amplifier, and, subsequently, multiple fluorescently tagged label probes bind to each amplifier, generating a tree-like structure on the target RNA molecule with a high number of associated fluorescent molecules. Studies have shown that the PrimeFlow^TM assay has high sensitivity, with a detection limit of ~10 copies/cell for mRNA and ~20 copies/cell for miRNA [5], while also known to be specific [1]. Currently, the available commercial kit can be multiplexed to detect four RNAs and multiple target proteins [1,4].

Further, when combined with cell immunophenotyping, the PrimeFlow^TM assay can provide valuable information on the presence of mRNA and protein in predefined cell populations in a tissue. Thus, we advanced the implementation of the PrimeFlow^TM approach for the detection of luciferase transgene mRNA and protein in cells from the spleens of mice treated with luciferase mRNA-lipid nanoparticles (LNPs) in vivo. We found that, despite challenging execution (e.g., identifying and qualifying compatible reagents, temperature requirements, multistep labor-intensive protocol) and the need for further optimization (e.g., cell loss), the PrimeFlow^TM assay can support simultaneous multiplex detection of transgene mRNA and protein in a cell type–specific manner in samples from preclinical studies with potential to provide otherwise unattainable information for development of RNA-based and other types of biotherapeutics.

Materials and reagents

Biological materials

1. Male BALB/c mice (8–10 weeks old, 25–30 g) (Charles River Labs)

Reagents

1. PrimeFlow^TM RNA Assay Kit (Thermo Fisher Scientific, catalog number: 88-18005-210), containing:

a. PrimeFlow^TM RNA Fixation Buffer 1A (catalog number: 00-18100-54)

b. PrimeFlow^TM RNA II Fixation Buffer 1B (catalog number: 00-18200-54)

c. PrimeFlow^TM RNA Permeabilization Buffer (10×) (catalog number: 00-18300-43)

d. PrimeFlow^TM RNA Fixation Buffer 2 (8×) (catalog number: 00-18400-53)

e. PrimeFlow^TM RNA Wash Buffer (catalog number: 00-19180-26)

f. PrimeFlow^TM RNA Target Probe Diluent (catalog number: 00-19185-12)

g. PrimeFlow^TM RNA PreAmp Mix (catalog number: 00-16000-53)

h. PrimeFlow^TM RNA Amp Mix (catalog number: 00-16001-53)

i. PrimeFlow^TM RNA Label Probe Diluent (catalog number: 00-19183-100)

j. PrimeFlow^TM RNase Inhibitors (100×) (catalog number: 00-16002-19)

k. PrimeFlow^TM Compensation Kit (catalog number: 88-17009-42)

l. IC Fixation Buffer (catalog number: 00-8222-758)

m. Label Probes, 100× (catalog number: 00-16003-32)

Note: Expiration dates and storage and handling instructions for all materials in the kit are provided by the manufacturer.

2. Antibodies

Note: All antibodies and antibody mixes were stored refrigerated and protected from light. Freezing and thawing were not examined and are not recommended.

a. Rat anti-mouse CD45-BUV496, clone 30-F11 [BD Biosciences (BD Horizon^TM), catalog number: 569673]

b. Rat anti-mouse Ly-6C-BUV805, clone HK1.4 [BD Biosciences (BD OptiBuild^TM), catalog number: 755202]

c. Mouse anti-mouse H2Kd/H2Dd (MHC-I)-BV421, clone 34-1-2S [BD Biosciences (BD Horizon^TM), catalog number: 567294]

d. Rat anti-mouse CD19-BV605, clone 6D5 (BioLegend, catalog number: 115540)

e. Mouse anti-mouse NK1.1-BV711, clone PK136 [BD Biosciences (BD Horizon^TM), catalog number: 569723]

Note: Immunolabeling of the NK1.1 marker cannot be employed with every mouse strain, as some strains are considered NK1.1-negative despite having an NK cell population. Strain-appropriate alternatives should be used.

f. Hamster anti-mouse CD3-BV750, clone 145-2C11 [BD Biosciences (BD OptiBuild^TM), catalog number: 746988]

g. Rabbit anti-firefly luciferase-PE, clone EPR17789 (Abcam, catalog number: AB237253)

h. Rat anti-mouse CD11b-APC-eFluor780, clone M1/70 [Thermo Fisher Scientific (eBioscience^TM), catalog number: 47-0112-82]

3. Target probe sets, stored frozen (-20 °C), as recommended by the manufacturer:

a. PrimeFlow^TM Type 4 Mouse β-Actin target probe set for Alexa Fluor 488 [Thermo Fisher Scientific (Invitrogen), catalog number: PF-210, VB4-10432]

b. PrimeFlow^TM Type 1 luciferase target probe set for Alexa Fluor 647 (custom-designed VPWCWFC Synthetic G6 Luc co) [Thermo Fisher Scientific (Invitrogen), catalog number: PF-210]

4. BD OMICS-Guard sample preservation buffer (BD Biosciences, catalog number: 570911), stored refrigerated

5. BD Pharmingen^TM stain buffer (BSA) (BD Biosciences, catalog number: 554657), stored refrigerated

6. BD Pharmingen^TM stain buffer (FBS) (BD Biosciences, catalog number: 554656), stored refrigerated

7. Water (nuclease-free) (Fisher BioReagents, catalog number: BP2484-50), stored at ambient temperature

8. VitaStain^TM AO/PI staining solution (Nexcelom Bioscience, catalog number: CS2-0106-5mL), stored refrigerated

Solutions

1. Permeabilization buffer (see Recipes)

2. Fixation buffer 2 (see Recipes)

3. RNA wash buffer with 1× RNase inhibitors (see Recipes)

Recipes

1. Permeabilization buffer

Reagent	Dilution	Final concentration
PrimeFlow^TM RNA permeabilization buffer (10×)	1:10	1×
PrimeFlow^TM RNase inhibitors (100×)	1:100	1×

Dilute in DNase/RNase-free water.

2. Fixation buffer 2

Reagent	Dilution	Final concentration
PrimeFlow^TM RNA fixation buffer 2 (8×)	1:8	1×
PrimeFlow^TM RNase inhibitors (100×)	1:100	1×

Dilute in PrimeFlow^TM RNA wash buffer.

3. RNA wash buffer with 1× RNase inhibitors

Reagent	Dilution	Final concentration in RNA wash buffer
PrimeFlow^TM RNase inhibitors (100×)	1:100	1×

Laboratory supplies

Note: Based on the information provided by the manufacturers (website description, certificate of analysis), the supplies listed below are considered RNase-free.

1. 5 mL polystyrene round-bottom tube (12 × 75 mm style) (FACS tube) (Falcon, catalog number: 352054)

2. V-bottom 96-well plate (Costar, Corning, catalog number: 3896); supplemented with a lid from a flat-bottom 96-well plate (Costar, Corning, catalog number: 3596)

3. 50 mL polypropylene conical tube (30 × 115 mm style) (Falcon, catalog number: 352070)

4. 15 mL high-clarity polypropylene conical tube (17 × 120 mm style) (Falcon, catalog number: 352096)

5. 40 μm cell strainer (filter) (Fisher Scientific, catalog number: 07-201-430)

Equipment

1. BD FACSymphony^TM A5 Cell Analyzer (BD Biosciences, catalog number: 660964; any flow cytometer with appropriate optical setup can be used)

2. 40 °C incubator (Quincy Labs, model: 10E)

3. ViewRNA^TM Temperature Validation kit (Thermo Fisher Scientific, catalog number: QV0523)

4. Centrifuge (Thermo Fisher Scientific Sorvall X4R Pro-MD, catalog number: 75009521)

5. Cellometer Vision Cell Profiler (Nexcelom, model: Cellometer Vision)

Software and datasets

1. BD FACSDiva^TM (BD Biosciences, version 9.1)

2. FlowJo^TM (BD Biosciences, version 10)

3. Prism [GraphPad (Dotmatics), version 10]

4. Excel (Microsoft)

Procedure

English

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文章信息

稿件历史记录

提交日期: Mar 17, 2026

接收日期: May 31, 2026

在线发布日期: Jun 15, 2026

出版日期: Jun 20, 2026

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如何引用

Gallolu Kankanamalage, S., Fan, X., Kundu, S., Villamizar, O., Roh, J. S., Cohen, J., Neubert, H. and Goihberg, P. (2026). PrimeFlow^TM Assay for Cell Type–Specific Co-detection of Transgene RNA and Protein in Mouse Spleens From Preclinical Studies. Bio-protocol 16(12): e5748. DOI: 10.21769/BioProtoc.5748.