Browse through PubMed and perform a search with keywords like transcriptomics, proteomics, genomics, sequencing or amplification. Pick any paper answering questions in cancer research, neurobiology, stem cell biology, immunology or developmental biology. Are you amazed by how often you come across the term “single-cell”? Don’t be!
The field of single-cell analysis has grown enormously in the recent years. Nature Methods has acknowledged the growing interest in the field by selecting “single-cell methods” as a Method to Watch in 2011 (1) and “single-cell sequencing” as The Method of the Year for 2013 (2). The demand for single-cell isolation technologies has simultaneously increased exponentially.
Pitfalls of bulk cell analysis
Every day you may walk into your cell culture laboratory, pick up your culture dish and harvest your cells to perform your planned assay. You would have cultured those cells based on the assumption that all the cells in the dish are identical and would only amplify your signal of interest and help you gain true insight. This does not always necessarily hold true. What remains cryptic and blurred are certain cell-to-cell distinctions or subtle differences at the single cell level that can result in significant variation in cellular behavior.
Conventional assays will only analyze the average responses from this bulk population of cells, masking the important information about small but potentially relevant subpopulations and blinding researchers to possibly interesting individual cell contributions. The complex heterogeneity of cells in culture poses a challenge for many experimental measurements, making it difficult to detect signal over noise. Therefore, enriched cell populations are becoming a prerequisite in order to let single cells tell life science and biomedical researchers what Petri dish cultures cannot.
Of single cells and isolation methods
The requirements for technologies to isolate single cells from diverse cell types and sources appear as heterogeneous as the purposes for which the cells are used downstream of the isolation process. As the selection criteria become complex, such as cells showing a chemical response, a morphological feature, fluorescence, antibiotic or drug resistance, or a combination of multiple parameters, sorting of target cells from a heterogeneous pool becomes technically challenging.
The performance and selection of a given single-cell isolation technology or instrument in turn is dictated by several needs: efficiency or throughput (how many cells can be isolated in how much time), purity (cells are free of contamination), cell viability (recovery of live cells after isolation), affordability (procurement, maintenance and running costs of the instrument), number of cells needed (minimum number to operate the device), yield (amount of material that can be extracted from one cell), space needed in the laboratory, ease of use and accessibility (for the instrument) and compatibility with existing workflows.
Currently available single-cell isolation technologies are typically classified based on the level of automation, distinguishing manual methods such as manual picking / micromanipulation and limiting dilution from automated devices such as flow cytometry / fluorescence-activated cell sorters (FACS), microfluidics-based systems and laser capture microdissection (LCM). Although these methods have their own advantages, they can have more than one drawback, either related to poor sensitivity, low throughput, high cost, lack of expertise, poor cell viability and integrity, low purity, less efficiency, less flexibility or broad sample nature and origin.
Technological advances on these fronts can only help unravel the biological complexity hidden within single cells. With QIAscout, picking cells just got a lot easier! The microraft array technology is now empowering every lab to do single-cell research by bringing the advantages of the available commercial methods to this efficient, affordable and accessible single-cell isolation device.
Going from Sample to Insight in single-cell biology
Remember, I discussed how compatibility of an instrument with the workflow that you design becomes a driving factor when choosing a certain instrument over another. QIAscout enables easier interfacing with various sample types upstream and multiple downstream analyses such as single-cell genomics, transcriptomics, sequencing and microRNA analysis.
Multiple sample types including cell suspensions, adherent cells, primary cells, dissociated cells, enriched cells and circulating tumor cells (CTCs) can be separated using the QIAscout.
Single-cell RNA-seq requires the successful combination of single-cell isolation techniques together with conversion of the minute amount of cellular RNA into cDNA followed by parallel sequencing of cDNA libraries. The QIAseq FX Single Cell RNA Library Kit is an end-to-end library preparation solution for RNA-seq from single cells. The kit is ideally suited for transcript discovery and differential expression from single eukaryotic cells.
Did you know that the QIAseq FX Single Cell DNA Library Kit provides a complete solution for whole genome sequencing from isolated single cells? The kit is ideally suited to the analysis of aneuploidy, copy number variation and sequence variation in single cells.
DNA sequence analysis and genotyping of biological samples using NGS platforms are often limited by the small amount of sample available. The REPLI-g Single Cell Kit is specially designed to uniformly amplify genomic DNA from single cells.
The REPLI-g WTA Single Cell Kit enables reliable investigation of effects on transcription regulation at the single-cell transcriptome level and allows uniform amplification of all transcripts from just single cells.
Whole transcriptome amplification (WTA) overcomes limited RNA availability for NGS-based gene expression analyses. Discover how the REPLI-g Single Cell RNA Library Kit allows reliable investigation of the transcriptome from a single cell with minimal bias. The kit offers an efficient, PCR-free method for RNA library construction from single cells for RNAseq applications on NGS platforms.
Similarly, conducting single-cell genomic analysis using NGS methods has traditionally been challenging since the amount of genomic DNA present in a single cell is very limited. The REPLI-g Single Cell DNA Library Kit enables accurate amplification of genomes with negligible sequence bias and minimal genomic drop-outs.
Until recently, the method of choice to study gene expression of single cells was multiplexed qRT-PCR and target detection was highly biased with a competitive effect between high- and low-abundance targets. Now with the launch of the new QuantiNova Multiplex RT-PCR Kit, researchers can gain more information even from single cells without losing low-abundance targets.
Are you performing whole miRNome profiling, disease/pathway-specific miRNA expression analysis, or individual miRNA quantification from a single cell? Take advantage of our miScript Single Cell qPCR Kit and go from a single cell or minute amounts of purified RNA to isolated and amplified miRNAs in a single, streamlined workflow.
Explore the power of one
To sum up, single-cell analysis now stands poised to unravel longstanding questions in life science and biomedical research. Considering the rapid progress in the development of single-cell isolation technology such as the QIAscout, many of the biological complexities underlying normal and diseased conditions will be elucidated at the single cell level.
This holiday season, we have granted your wish! Learn how QIAscout can help you unlock the mysteries of your single cell.