Encouraged by the reproducibility and ease of use of this protocol for analyzing a few hundred cells, we next adapted this method for expression analysis of cells growing in soft agar. Anchorage-independent growth in soft agar is the most accurate and stringent in vitro assay for detecting malignant transformation of cells (15). For instance, introduction of a defined set of genetic lesions transforms primary human cells into cells capable of growing in soft agar and initiating tumors in nude mice (16, 17). Strikingly, even in a genetically-defined population only a small percentage of the cells form colonies, suggesting that additional characteristics are selected for in soft agar (18). Transcriptional analysis of such colonies growing in soft agar to identify characteristics of this subpopulation is precluded by the small size of individual colonies, whereas total recovery of cells from the soft agar blends the cells that have grown into a colony with the majority of cells that have not. To enable monitoring of transcript levels, individual colonies consisting of 100 to 300 cells were picked from molecular biology grade soft agarose (low gelling agarose, A9045; Sigma-Aldrich, St. Louis, MO, USA) using a 200 µL pipette tip guided by a stereomicroscope and each colony was liberated from the agarose by centrifugation and resuspended in six µl lysis buffer. We found that when using high-grade agarose there was no apparent inhibition of subsequent enzymatic steps (data not shown). Unlike single cells sorted by FACS, disruption of soft agar colonies requires an additional round of sonication, or three rapid freeze-thaw cycles for efficient lysis and consistent signals (Figure 3A). For ease of use, we proceeded to use rapid freeze-thawing in this protocol.
Human mammary epithelial cells (HMECs), after the introduction of hTERT, SV40 small t, and active Ras remain limited in their growth in soft agar by the activation of the p53 pathway (16). Polyclonal transduction with retroviruses expressing shRNAs targeting either p53 directly or p14ARF (a classical upstream activator of p53) results in colony formation. When tested on the polyclonal bulk population (by lysing 10,000 cells in 140 µL lysis buffer, and using 7 µL in our direct RT for an equivalent of 500 cells), p53 shRNAs caused an efficient reduction in the mRNA levels of two direct transcriptional targets of p53: p21 and Hdm2 (19). However the p14ARF shRNA showed only a modest reduction in p53 activity as measured by the expression of its target genes p21 and Hdm2 (Figure 3B, solid symbols). This was surprising since the p14ARF shRNA-transduced population is equally potent in generating soft agar colonies as the p53 shRNA cells (data not shown). One explanation could be that colonies arise from a subset of the population that has been most efficiently transduced with from the p14ARF shRNA. Indeed, when testing p53 activity through its target genes in six independent colonies, there is no statistically significant difference in p21 and Hdm2 expression levels between cells with p53- or p14ARF-shRNAs (Figure 3B, open symbols), supporting the hypothesis that only p14ARF-knockdown cells that effectively prevented p53 activation were able to grow in soft agar. Notably, colonies with the strongest reduction in p21 mRNA levels also had the strongest reduction in Hdm2 mRNA levels, indicating variation between individual colonies represents biological variation in the extent of p53 inactivation rather than technical variation.
In summary, we describe a convenient method that can be used to directly test hypotheses regarding the transcriptional response of a cellular subset, without the need for RNA amplification or large-scale single cell analyses. The direct RT method on soft agar colonies allows the detection of relevant expression levels in the cells that form colonies and reveals selection events that are obscured in the bulk population.
We would like to thank Manu Beillard for valuable input. This research was supported by the Singapore Ministry of Health's National Medical Research Council under its Exploratory Development Grant (NMRC/EDG/0066/2009) and Individual Research Grant (NMRC/GMS/1250/2010) scheme.
The authors declare they have no competing interests.
Address correspondence to Alexandra M. Pietersen, Laboratory of Mammary Gland Biology, National Cancer Centre Singapore, 11 Hospital Dr, 169610, Singapore. E-mail: [email protected]
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