Considerations When Planning Single Cell RNA Sequencing Analysis

RNA sequencing, also known as RNA-seq, is a genomic method for finding and quantifying messenger and micro RNA molecules in a biological sample. This method is useful for studying how cells respond to certain environmental, medical, and/or chemical factors to which they are exposed. In recent years, RNA-seq has contributed significantly to medical discovery and innovation. The procedure is typically carried out on samples containing thousands to millions of cells in order to ensure that there are enough RNA molecules present for accurate and statistically significant results. Due to the method’s popularity and flexibility it is not a surprise that interest has grown in studying the RNA profiles of single cells. There has been a significant amount of progress made in the field of single-cell RNA-sequencing (scRNA-seq) since the first study on the topic was published in 2009. Rapid ongoing maturation of bioinformatics methods and the increasing commercial availability of scRNA-seq platforms now allows researchers to utilize this technique as a compliment to other methods of RNA sequencing.

Examples of scRNA-seq and multi-cell/bulk (sourced from multiple cells or bulk tissue sample) RNA profiling complementing each other can be found across numerous scientific research disciplines. One recent study by Ruiz M, et al. (2022), demonstrates this complementarity as collaborators aimed to study cultivated limbal epithelial cells (cLECs) in order to understand how to prevent and treat limbal stem cell deficiency (LSCD) disease. In the study, they examined not only at the microRNA (miRNA) profile of the single cLECs but also at the extracellular bulk miRNAs expression levels. The extracellular miRNA expression levels were measured using an innovative extraction-free gene expression profiling (GEP) technology. The bulk miRNA assay simultaneously quantified 2,083 human miRNA transcripts. By doing both single cell and bulk miRNA sequencing, the researchers were able to more accurately identify a specific miRNA that can serve as a biomarker for early detection and treatment efficacy indicator for patients suffering from LSCD.

In another instance, Salome B, et al. (2022) aimed to characterize the role of immune checkpoint molecules in bladder cancer, which prevent tumor escape mechanism from furthering metastasis. To better understand the tumor microenvironment (TME) the researchers performed single-cell mRNA sequencing on fresh bladder tumor samples. To corroborate their scRNA-seq findings the researchers also used ultra-efficient multi-cell mRNA sequencing technology which profiled FFPE sections of the same bladder tumor samples. The bulk mRNA assay used for this study interrogated 2,549 genes associated with tumor biology. By analyzing the results of both scRNA-seq and bulk RNA-seq the researchers concluded that certain biomarkers can be used as indicators of restoration of cells’ anti-tumor functions.

In this final example, Johnson K, et al. (2022) set out to obtain comprehensive miRNA profiles of patients at different stages of non-alcoholic fatty liver disease (NAFLD). The scRNA-seq data showed that there is a difference in expression levels of certain biomarkers in cirrhotic compared to uninjured liver cells. Blood samples from the same patients were processed using whole transcriptome multi-cell miRNA profiling as well. The researchers used these complementary methods to demonstrate an unbiased global profile of circulating miRNAs and identify a number of differentially expressed genes which can act as liver disease biomarkers.

The ability to study thousands of cells in a single run has facilitated prospective studies of highly heterogeneous clinical samples. This can have a profound impact on both translational applications as well as our understanding of basic tissue architecture and physiology. With increasing opportunities for single-cell transcriptome characterization, we have witnessed remarkable diversification of experimental protocols as well. There are many other examples of single-cell and bulk RNA transcriptomics working hand in hand.

Bulk RNA sequencing is essential for studies that look at comprehensive transcriptomic profiles, interrogate homogeneous systems, confirm the findings of single cell RNA sequencing analysis, identify novel biomarkers for specific diseases, and/or look for effects of drugs on gene expression profiles during early stages of drug development. On the other hand, single cell RNA sequencing can be used to study separate cell populations and/or understand cell population dynamics.

Perhaps, the “Fruit Salad” anecdote from the Cancer Research UK Cambridge Institute explains the complementarity of the two methods the best.


All in all, a comprehensive approach to RNA sequencing is much better suited for biomedical research. Looking at RNA expression within a single cell has advantages and limitations to be considered. When working with biological material the opportunity to perform bulk RNA analysis should not be overlooked as it may uncover novel insight in the form of potential biomarkers or at a minimum helping to confirm observations from the single cell assay. Having clearly defined biological objectives and a rational experimental design are often vital for making an informed decision about the optimal approach.

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Page last updated November 01, 2022