TY - JOUR
T1 - Microscale Gene Expression Analysis of Tumor-Associated Macrophages
AU - Attri, Kuldeep S.
AU - Mehla, Kamiya
AU - Shukla, Surendra K.
AU - Singh, Pankaj K.
N1 - Funding Information:
This work was supported in part by funding from the National Institutes of Health grant (R01 CA163649, R01 CA210439, and R01 CA216853, NCI) to PKS; the Specialized Programs of Research Excellence (SPORE, 2P50 CA127297, NCI) to PKS. We would also like to acknowledge the Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727, NCI) for supporting shared resources. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - Macrophages, apart from being the key effector cells of the innate immune system, also play critical roles during the development and progression of various complex diseases, including cancer. Tumor-associated macrophages, infiltrate tumors during different stages of cancer progression to regulate motility, invasion, and intravasation to metastatic sites. Macrophages can exist in different polarization states associated with unique function in tumors. Since tumor-associated macrophages constitute a very small proportion of tumor cells, analysis of gene expression pattern using normal extraction buffer-based methods remains a challenging task. Therefore, it is imperative to develop low-throughput strategies to investigate transcriptional regulations from a small number of immune cells. Here, we describe an efficient, sensitive, and cost-effective approach for gene expression analysis of a small number of fluorescence-activated sorted tumor-associated macrophages. Our analyses from the different number of stable, primary, and sorted macrophages suggest 5,000 cells is an optimal number for performing quantitative, real-time PCR analysis of multiple genes. Our studies could detect expression of macrophage-specific genes from cultured primary macrophages, and FACS-sorted macrophages from different biological tissues without introducing biases in comparative gene expression ratios. In conclusion, our kit-based method for quantitative gene expression analysis from a small number of cells found in biological tissues will provide an opportunity to study cell-specific, transcriptional changes.
AB - Macrophages, apart from being the key effector cells of the innate immune system, also play critical roles during the development and progression of various complex diseases, including cancer. Tumor-associated macrophages, infiltrate tumors during different stages of cancer progression to regulate motility, invasion, and intravasation to metastatic sites. Macrophages can exist in different polarization states associated with unique function in tumors. Since tumor-associated macrophages constitute a very small proportion of tumor cells, analysis of gene expression pattern using normal extraction buffer-based methods remains a challenging task. Therefore, it is imperative to develop low-throughput strategies to investigate transcriptional regulations from a small number of immune cells. Here, we describe an efficient, sensitive, and cost-effective approach for gene expression analysis of a small number of fluorescence-activated sorted tumor-associated macrophages. Our analyses from the different number of stable, primary, and sorted macrophages suggest 5,000 cells is an optimal number for performing quantitative, real-time PCR analysis of multiple genes. Our studies could detect expression of macrophage-specific genes from cultured primary macrophages, and FACS-sorted macrophages from different biological tissues without introducing biases in comparative gene expression ratios. In conclusion, our kit-based method for quantitative gene expression analysis from a small number of cells found in biological tissues will provide an opportunity to study cell-specific, transcriptional changes.
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U2 - 10.1038/s41598-018-20820-4
DO - 10.1038/s41598-018-20820-4
M3 - Article
C2 - 29402936
AN - SCOPUS:85041688620
SN - 2045-2322
VL - 8
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 2408
ER -