Determination of absolute expression profiles using multiplexed miRNA analysis

Abstract

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR’s ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes.

Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes

“…The metabolic substrate-based studies described in this paper depend on the ability to introduce high levels of flux into the sialic acid biosynthetic pathway with negligible cytotoxicity and minimal growth inhibition. We previously achieved these objectives in other human (and rodent) cells using ‘high-flux’ 1,3,4-O-butanoylated ManNAc analogs [ 40 , 46 , 70 , 71 ] but the cytotoxicity of the newly-synthesized, alkyne-modified analog 1,3,4-O-Bu 3 ManAl was unknown; moreover, the three cell lines used in this study (MCF10A, T-47D and MDA-MB-231) had not been comparatively screened with these analogs in ‘assay media’ where the cells were grown in low serum (1.0%; to prevent recycling of sialic acids from glycoproteins contained in serum [ 57 ]) as well as without antibiotics (to avoid inhibition of sialylation [ 59 ]). Therefore, as a prelude to subsequent metabolic profiling, we first evaluated cell viability by measuring proliferation after treatment with 0, 10, 100, or 250 μM of 1,3,4-O-Bu 3 ManNAc (Panel A in S2 Fig ), 1,3,4-O-Bu 3 ManNAz (Panel B in S2 Fig ), or 1,3,4-O-Bu 3 ManNAl (Panel C in S2 Fig ) for 6, 24, or 48 h and observed no statistically-significant differences between the control and test cells.…”

Quantitative and time-resolved miRNA pattern of early human T cell activation

“…These studies report a total miRNA content within the magnitude of ∼10 5 molecules/cell ( 87–89 ). Likewise, the overall range of the most abundant miRNAs (∼10 3 –10 5 molecules/cell) is according with a former quantification approach of human cell lines ( 90 ). Moreover, the finding of only a limited number of highly abundant miRNAs within specific cell types is consistent with the literature ( 88 , 91 ).…”

Visible-light triggered templated ligation on surface using furan-modified PNAs

“…miR-29, miR-21, miR-17, miR-16a and miR-15a). 68 From the results previously obtained in solution, the reactivity of the system seemed to follow the nucleophilicity of the probe, with hydrazine probes showing higher performance as compared to amine probes. Moving to surface experiments, this behavior is not uniformly maintained, yet seems more complex.…”