From Cultures to Results in One Day: A New Workflow for Accurate and Reproducible Large Scale qPCR Expression Analysis from Limited Samples in Stem Cell Research

Mark Kibschull1, Stephen Lye1,2,3, Steven Okino4, Haya Sarras5 1Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada;  2Departments of OBS/GYN, Physiology, and Medicine, University of Toronto, Toronto, Canada;  3Fraser Mustard Institute for Human Development, University of Toronto, Toronto, Canada;  4Gene Expression Division, Life Science Group, Bio-Rad Laboratories, Hercules, United States;  5Gene Expression Division, Life Science Group, Bio-Rad Laboratories, Mississauga, Canada

Abstract
Several applications in the stem cell field use RT-qPCR to routinely assess the quality of cell cultures by analyzing specific sets of marker genes. For instance, during lineage differentiation of human embryonic stem cells (hESC) into defined cell types, or, vice versa during reprogramming of somatic cells into induced pluripotent stem cells (iPSC), researchers need to assess the performance of their culture systems frequently. Often, only a small sample of a stem cell culture can be collected without compromising long-term experiments. Although RT-qPCR is highly sensitive, a small sample sizes may restrict the number of targets that can be analyzed, and amplification steps that are applied to increase starting material may introduce bias in gene expression quantification.
Here, we present a 4-step workflow using validated reagents, allowing the (i) direct, single-step generation of gDNA-free total RNA lysates from micro samples of hPSC colony biopsies or individual embryoid bodies. The lysate is fully compatible with (ii) reverse transcription reagents to produce cDNA. In a (iii) target-specific multiplex pre-amplification reaction, using validated primers, 100 cDNA targets are 1000 fold enriched, and thereby providing sufficient material for subsequent (iv) SYBR-Green based qPCR analysis. We show that this workflow allows fast and reproducible gene-expression profiling, and introduces only minimal bias (<0.75 Cq values) during the pre-amplification step, when data are compared to the non-amplified starting cDNA samples. When comparing individual samples of the hESC line CA1, collected and processed at different time points, and consisting of varying sample sizes (500-50,000 cells), this workflow delivers excellent and unbiased correlations in normalized gene-expression data. We demonstrate that during iPSC derivation, individual colonies can now be analyzed for hundreds of different markers during the reprogramming process, by using only small clusters of cells collected from culture plates. Also, during long-term embryoid body (EB) culture experiments the differentiation of specific cell types can be monitored by lysing and processing as little as individual EBs. In conclusion, this workflow allows accurate transcriptional profiling of micro-samples for hundreds of targets using SYBR-Green-based RT-qPCR analysis within one work day. Therefore, it provides a rapid, more cost-effective method to screen and evaluate various types of stem cell cultures without compromising continuing experiments. This allows researchers not only to reduce individual culture formats, costs and processing time, but also to increase sample throughputs and generate reliable gene-expression data from limited or precious biological material.