Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages.
MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best-suited approach for the functional analysis of M2 macrophages.
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-5946, USA.
Yee Seng
YS
Ng
Hernan
H
Roca
David
D
Fuller
Sudha
S
Sud
Kenneth J
KJ
Pienta
D002232:Carbocyanines; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002470:Cell Survival; D002478:Cells, Cultured; D018274:Electroporation; D005456:Fluorescent Dyes; D006801:Humans; D008264:Macrophages / Q000145:classification / Q000378:metabolism* / Q000473:pathology; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D009000:Monocytes / Q000378:metabolism / Q000473:pathology; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics; D014162:Transfection / Q000379:methods; D020298:U937 Cells
D002232:Carbocyanines; D005456:Fluorescent Dyes; D035683:MicroRNAs; D012322:RNA Precursors; C089187:cyanine dye 3