Single-Cell Challenges - GEN article

Utilizing qPCR for single-cell analysis remains a significant hurdle, according to Martina Reiter, Ph.D., CEO of BioEPS. “Although single-cell analysis is not new, we still don’t have a final solution as to how to handle it. The biggest problem is technical variance during the experimental steps, especially in sampling analysis and subsequently data analysis.”
“Often samples are generated from laser capture microdissection and flow cytometry sorting. Sample amounts are very small and variances are huge. When you don’t pay attention in the beginning to such variances, downstream processes are even more inaccurate.”
What about solutions? “We are at the beginning of working on this and are continuing to determine the right way to do it,” Dr. Reiter said. “Substantial improvements in the analysis steps are needed. You cannot use standard analysis methods; they need to be modified for single-cell qPCR. But it is still difficult to modify so that technical variances are reduced as much as possible. Despite this, qPCR technology is sensitive enough to detect specific genes out of one cell.”
The MIQE guidelines are a newly proposed set of suggestions to standardize and document qPCR. “MIQE does not cover specific single-cell analysis so far, and qPCR advances need to be made in documenting and determining RNA integrity, in how to handle the necessity of preamplification, in the ability to more accurately sample cells, and in downstream analysis methodologies.”
Recognizing the importance of tackling these issues now will especially impact the future of therapeutics. “Ultimately, these problems will be solved,” Dr. Reiter predicted. “For the present, we need to realize that while qPCR for single-cell analysis is possible, especially technically, the larger issues of experimental design and variation remain and should be addressed.”
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