Gene expression profiling is a powerful method that allows one to relate the expression of specific genes to a particular cellular phenotype. For example, when comparing cancer tissue to healthy tissues within an organ.
However, gene expression patterns often vary even among cells within the same tissue sample. Traditional methods of gene expression analysis on bulk samples do not allow researchers to distinguish the contributing variation from individual cells.
Gene expression analyses performed on single cells can offer a powerful method to resolve sample heterogeneity and reveal hidden biology in multicellular tissue samples. This approach allows in-depth analysis of complex biological phenomena such as cancer, development, and microbial ecology.
Described below is a complete workflow of single cell gene expression analysis offered by BD.
To learn more about individual stages of the process and solutions offered by the company, visit the BD webpage.
Isolation of Single Cells
The first step in a workflow of single cell gene expression analysis is isolation of single cells from a sample. Single cells or small numbers of cells can be isolated and prepared by limiting dilutions, laser capture micro-dissection, microfluidics technologies, or fluorescence-activated cell sorting (FACSTM).
FACS has emerged as a leading platform for this purpose because it is fast, enables high-throughput processing of a cell mixture, and ensures the delivery of single cells or a small number of cells into a chosen receptacle at a very high purity level.
Prior to sorting the cells, samples can be stained with fluorescent antibodies specific to cell surface markers or intracellular marker proteins, or nucleic acid dyes. This makes it is possible to sort live cells, as well as different cell types, based on the expression levels of target cell markers.
Figure 2 shows how cell sorting is performed in a BD FACS sorting instrument.
A FACS instrument incorporates the fluidic, electronic, and optical systems of a flow cytometer.
When a sample is introduced into a FACS sorter, the fluidic system funnels the cells into a single stream so that they pass one at a time through a laser beam or a series of laser beams.
When each cell passes through the laser beam(s), it scatters light and may emit fluorescence light based on cell surface or intracellular fluorescent staining. Light scatter and fluorescence signals are collected and detected, and a decision is made whether the cell is of interest and should be sorted.
Flow cytometry cell sorters can collect the cells into collection tubes, trays, and slides, as well as 96-well or 384-well tissue culture and PCR plates.
BD makes a variety of instruments for sorting single cells to fit the researcher’s experimental need, the lab’s budget, and the operator’s flow sorting experience and preference.
To learn more about FACS sorters offered by BD, visit the BD Biosciences website.
Generation of gene expression sequencing libraries
To obtain sequencing information from low input samples, such as single cells, amplification of the sequencing library is required. At this stage, it is important to control for the introduction of artefacts, such as amplification bias.
Molecular barcoding is an integral part of BDTM Precise assays - ultra-sensitive RNA sequencing assays designed to examine large numbers of low input mRNA samples in a high-throughput manner. To improve workflow and mitigate amplification bias, these assays append three layers of DNA barcoding to each mRNA molecule.
- A molecule-specific barcode (Molecular Index) is used to identify the molecule of origin of each sequenced read in an amplified sequencing library. This provides a more accurate representation of transcript abundance in the original sample prior to amplification.
- Well-specific barcodes are used to link individual molecules from the same single cell. They also allow for pooling of individual cells to simplify the library preparation protocol.
- Plate-specific barcodes are used to allow content from multiple plates to be run in a single sequencing run.
Custom BDTM Precise Targeted assays can be designed to interrogate approximately 80-240 genes simultaneously. Pre-designed panels are also available, covering specific biological areas, such as breast cancer, regulatory T cells, prostate cancer and other cell types.
In a BDTM Precise whole transcriptome analysis (WTA) assay, any transcript with a poly A tail will be interrogated. This assay best suited for gene expression discovery studies.
BD has developed a series of BDTM Precise targeted assays, and a WTA assay to fit the specific needs of researchers.
To learn more about BDTM Precise kits, visit the BD website.
The BDTM Precise assay sequencing data analysis pipeline is provided via Seven Bridges Genomics, a secure online cloud environment.
The pipeline takes FASTQ files from the sequencing run and collapses molecular indices using a proprietary algorithm. The number of molecules per gene per cell is reported in a Microsoft® Excel® (or tabular) file, which can then be imported into statistical software packages such as R for secondary data analysis.
Single cell sorting experiment
The following experiment demonstrates the data, which can be obtained in BD single cell analysis workflow.
Peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood and stained with an antibody cocktail containing the cell surface markers CD16 PE, CD56 PE, CD3 FITC, CD45 PerCP-CyTM5.5, CD4 PerCP-CyTM7, CD8 APC-Cy7, and CD19 APC (BD MultitestTM 6-color TBNK Reagent, Cat. No. 644611).
Pan T cells were identified by CD45+CD3+ staining; B cells were identified by CD45+CD3-CD19+; and NK cells were identified by CD45+CD3-CD16/CD56+.
Using BD FACS sorter, thirty cells from each of the T, B, and NK populations were sorted into each BDTM Precise plate.
For the single T, B, and NK cells sorted from this experiment, mRNA expression was measured by the BDTM Precise targeted assay using a panel targeting 100+ genes expressed by immune cells in human peripheral blood (Figure 6). Genes included those that encode surface proteins, cytokines, and transcription factors.
Once the transcript counts are obtained from the automated data analysis pipeline, the sequencing data can be further analyzed by various methods for biological relevance.
The following figure links mRNA expression measured by the BDTM Precise assay with protein expression measured by antibody staining.
The combined workflow of BD FACS sorting combined with BDTM Precise assays provides investigators with a complete solution for high-throughput single-cell gene expression sequencing analysis.
To learn more about the offer, visit the BD website.
Publication: A Complete Workflow from Single Cell Isolation to mRNA Sequencing Analysis. Wang, A et al. (White paper by BD). Click here to view.