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Protein Validation of Single-Cell RNA Sequencing (RNA-Seq) Data

Do you have a list of targets from your single-cell RNA sequencing (RNA-seq) experiments and want to verify protein expression levels for your targets? Only Single-Cell Westerns on Milo™ offer the versatility to validate diverse protein targets discovered in your sequencing runs.

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Single-cell western instrument Milo

Milo Validates Single-Cell RNA-Seq Data

Milo provides single-cell protein expression information to validate your single-cell RNA data. Since mRNA levels do not always correlate with functional protein levels, pairing single-cell RNA data with single-cell protein expression data is critical to making accurate and complete conclusions about cellular function. Milo uses the large Western catalog of antibodies & can easily measure proteins irrespective of their location in or on a cell, making Milo the only platform with the versatility to detect diverse protein targets that are discovered in your RNA-sequencing runs.

  •  Validate RNA heterogeneity data
  •  Quantify protein heterogeneity
  •  Confirm gene expression results
  •  Correlate RNA-seq data to protein expression on a single-cell level

Single-Cell Westerns for Protein Expression Heterogeneity Analysis

The Milo Single-Cell Western platform provides scientists with protein validation data for their single-cell RNA-seq gene expression data (FIGURE 1). Milo users can measure protein expression levels in over 1,000 individual cells per run and can multiplex with typical assays detecting approximately four proteins per cell simultaneously using a variety of multiplexing approaches.

Regardless of what targets are uncovered in your RNA-seq run, Milo has the flexibility and versatility to detect them at the protein level. Milo uses commercially available Western blot antibodies, giving users access to the broadest set of detection reagents to validate even uncommon targets that emerge from their sequencing runs. Furthermore, users can measure proteins irrespective of where they are located in or on the cell, measuring transcription factors and protein isoforms which can be challenging to measure by flow cytometry. Single-Cell Westerns can also be used to study post-translational modifications such as phosphorylation that are not revealed by RNA-seq analysis.

    Milo protein validation workflow of single-cell RNA seq data

    FIGURE 1. Workflow for protein validation of single-cell RNA-Seq data.


    HIF-1α mRNA and Protein Levels Do Not Always Correlate

    Proteins can be rapidly degraded after they are translated, leading to significant differences in the levels of mRNA versus functional protein within a cell. Expression of the transcription factor known as Hypoxia inducible factor 1-alpha (HIF-1α) is a classic example. HIF-1α mRNA and protein are constitutively expressed in cells. However, under normoxic conditions (when O2 is readily available) HIF-1α is rapidly ubiquitinated, targeting it to the proteasome for degradation (FIGURE 2). This process is mediated by an oxygen-dependent prolyl hydroxylase (PHD) and an E3 ubiquitin ligase known as von Hippel-Lindau (VHL) protein.

    Under hypoxic conditions (when O2 is scarce), PHD activity is inhibited and HIF-1α escapes proteosomal degradation. HIF-1α heterodimerizes with HIF-1β to form a transcriptionally-active complex that regulates the expression of >60 genes including vascular endothelial growth factor (VEGF) and erythropoietin (EPO), signaling molecules important for increasing O2 delivery to hypoxic tissues. As a result, HIF-1α mRNA and protein levels only correlate under hypoxic conditions but differ substantially under normoxic conditions.

    HIF-1a Protein Regulation
    FIGURE 2. HIF-1α protein regulation.

    Cellular Subpopulations Identified by Single-Cell Westerns

    RNA-seq analysis of the HeLa cell mixture showed that 100% of the cells expressed HIF-1α mRNA (FIGURE 3). However, Single-cell Western analysis with Milo revealed that only 46% of the cells identified by β-tubulin expression stained positive for HIF-1α protein (FIGURE 4). As expected, HIF-1α protein expression was degraded in the population of cells exposed to normoxic conditions. Single-cell protein expression did not correlate with single-cell RNA expression, highlighting the need for both protein and RNA expression information in single-cell gene expression studies.

    The single-cell RNA-seq data also identified two distinct cell populations within HIF-1α expressing cells based on differential expression of genes other than HIF-1α (FIGURE 5), uncovering other genes that may play a key role in hypoxic cellular processes. Milo allows researchers to validate these additional RNA targets revealed by single-cell RNA-seq experiments with protein expression data which may provide critical insights into the role these genes play in cellular function.

    Single-cell RNA-seq analysis plot of HIF-1α



    FIGURE 3. Single-cell RNA-seq analysis of HIF-1α. A mixture of DFO-treated and untreated HeLa cells was assayed for expression of HIF-1α. 100% of the cells were found to express HIF-1α RNA. Cell clustering based on β-tubulin expression revealed a single, homogeneous population.



    FIGURE 4. Single-Cell Western analysis of HIF-1α. A) HIF-1α protein was detected in only a subset of HeLa cells as identified by β-tubulin expression. Example separation images of six cells are shown with HIF-1α protein visualized in the 647 channel and β-tubulin visualized in the 488 channel. Green electrophoresis lanes indicate lanes where peaks were identified by Scout Software, whereas blue lanes indicate lanes with no peaks. Fluorescence intensity plots generated by Scout Software are shown on the right for all β-tubulin+ lanes. B) Bivariate analysis revealed that ~46% of analyzed β-tubulin+ cells were HIF-1α+.

    HIF1-a analysis with single cell western - Milo
    RNA sequences two populations



    FIGURE 5. RNA-seq analysis revealed two distinct cell populations. A heat map of gene expression shows that the mixture of DFO-treated and untreated HeLa cells contained two populations with distinct gene signatures. Both populations were positive for HIF-1α mRNA. Milo can be used to validate protein expression for these target candidates to understand their functional role in cellular function.