Profiling the molecular environment for single cells has become a valuable tool for not only understanding daily metabolic functions of cells, but also an integral part of elucidating the heterogeneous nature of diseases like cancer. As such, improving the sensitive parameters of this technique is essential for advancing a variety of research endeavors. Now, a team of investigators at the Ludwig-Maximilians University (LMU) in Germany has painstakingly optimized new methodology to improve single-cell RNA sequencing (scRNA-seq) protocols. Findings from the new study were published recently in Nature Communications through an article titled “Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq.”

“Single-cell technologies are already revolutionizing biology,” noted senior study investigator Wolfgang Enard, Ph.D., professor of molecular biology at LMU.

The human body is made up of on the order of 13 billion cells—and each of them has a distinct molecular profile. Even cells in the same tissue can differ, often subtly, from one another, and their activities can vary over time. Thus, single-cell analyses provide such a powerful tool for the characterization of cellular heterogeneities and the complex mechanisms that account for them.

Single-cell RNA sequencing makes it possible to obtain a snapshot of the functional state of any given cell—a molecular fingerprint, as it were. Essentially, the technique determines the composition of the mRNA population present in a cell. The inventory of the mRNAs present in a cell amounts to a list of the proteins made by that cell, which essentially reveals its functional state. By identifying the genes that were active at the time of analysis, it can tell us how these genes are regulated, and what happens when this process is disrupted by infection or other disease states.

Sequencing all the mRNAs from a single cell is a daunting task, and several different procedures have been designed and implemented—all of which begin with the “reverse transcription” of the isolated mRNAs into DNA by enzymes known as reverse transcriptases. The DNA copies are then replicated (“amplified”) and subjected to sequence analysis. Dr. Enard and his colleagues have now systematically modified one of these methods: single-cell RNA barcoding (SCRB-seq), significantly increasing its sensitivity.

In the current study, the research team set out to optimize the efficiency and sensitivity of SCRB-seq by utilizing a molecular crowding technique.

“We systematically evaluated experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis,” the authors wrote. “Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to develop a scRNA-seq library protocol we call molecular crowding SCRB-seq (mcSCRB-seq), which we show to be one of the most sensitive, efficient, and flexible scRNA-seq methods to date.”

“The trick is to supplement the reverse transcriptase reaction with an agent that increases the density of the medium. This induces molecular crowding, and speeds up the reaction so that more RNA molecules are transcribed into DNA strands,” Dr. Enard explained. A second modification reduces the incidence of preferential amplification of certain DNAs, which would otherwise distort the representation of the different RNAs present in the original cell. “Together, these modifications make our method, mcSCRB-seq, one of the most effective and economical RNA-seq procedures currently available,” Dr. Enard concluded.