This page contains a Flash digital edition of a book.
AL


Single-cell resolution of mRNA expression is a powerful tool for studying heterogeneous cell populations.


SmartFlare probes are nontoxic and do not alter gene expression, transla- tion or cell proliferation (Figure 2). The lyophilized probes are reconstituted with sterile nuclease-free water, diluted with phosphate-buffered saline (PBS) to the desired concentration, added to the cell culture medium and incubated with the cells overnight. RNA levels are typically detected using fluorescence microscopy or flow cytometry the next day.


Uptake, scramble and housekeeping control probes When screening live cells for RNA detection, it is important to include


three experimental control probes: uptake, scramble and housekeeping. The uptake control is always fluorescing and helps determine the cell’s ability to internalize the probes. It is important to use because not all cells will internalize the probes equally.


The scramble probe serves as a background control. Because the “re- porter” sequence attached to the scramble control has no counterpart in the genome of mammalian or eukaryotic cells, any fluorescence re- leased from the scramble control is nonspecific (e.g., probe degradation, incomplete quenching) and can serve as the background fluorescence. The housekeeping gene probe (e.g., GAPDH, β-actin) acts as a positive control that the SmartFlare probes can detect specific gene expression in a given cell population.


Heterogeneous cancer cell populations The heterogeneity of cancer cells is a significant barrier to developing


and receiving effective treatments. When studying mRNA expression levels in heterogeneous populations of cells, significant differences in data are expected if RNA is analyzed at the population level versus the single-cell level.


Quantitative RT-PCR cannot distinguish between high- and low- expressing subpopulations in heterogeneous cell samples. MCF-7 and MDA-MB-231 breast cancer cells have no/low and high expression levels of IL-6, respectively. The RNA expression profile changes accordingly when different percentages of these cells are mixed. While analyzing un- known heterogeneous cell populations, the researcher does not know whether variations in the curve represent a small subpopulation of cells changing expression to a very high degree, or if all the cells are changing expression to a very low degree collectively. Use of SmartFlare probes resolves expression at the single-cell level, allowing for the identification by flow cytometry of divergent cell populations not detectable by qRT- PCR. Therefore, single-cell resolution of mRNA expression is a powerful tool for studying heterogeneous cell populations.


AMERICAN LABORATORY 33 MARCH 2016


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60