Biology Reference
In-Depth Information
are beyond the scope of this chapter; however, detailed descriptions of next-
generation sequencing platforms and software for data analysis can be found in
Brown (2013) or Zhang et al. (2011) .
5.1.2 Upregulating micro-RNAs: Transfections with precursor
premiRNAs
Transfection of double-stranded pre-miRNAs into cells provides supporting data to
validate miRNA targets. These experiments can be carried out in six well plates with
cells plated at a density of 150,000-250,000 cells per well. Transfections are done
using Pre-miR
miRNA Precursor Starter Kit (Ambion Life Technologies, Grand
Island, NY) according to the manufacturer's instructions. After 24 h, the medium is
replaced with fresh medium containing DMSO for control cells (treated with a scram-
bledmicro-RNAsequence) ormediumwithdrug.Weuse siPort
Neo Fx (AmbionLife
Technologies, Grand Island, NY) and Optimem medium (ATCC) for the initial trans-
fection reactions with the pre-hsa-miRNA (experimental or negative control scrambled
sequence). For drug treatment experiments, we incubate at 37 Cand5%CO 2 for 24 h
and then replace the medium with the appropriate fresh medium. After 24 h, samples
can be collected for experimental assays (PCR, Western blotting, etc.).
5.1.3 Measuring activity of micro-RNAs
5.1.3.1 Measuring micro-RNA targets
Several databases are available to assist with the identification of potential and
known targets for micro-RNAs (e.g., TargetScan.org, microrna.org, mirdb.org).
Measurement of changes in these targets that correlate with levels of micro-RNAs
under experimental conditions provides supporting data for changes in micro-
RNA activity. For example, we found that mir-200c, a tumor suppressor, is upregu-
lated in MDA-MB-231 breast cancer cells in response to 40-nM paclitaxel treatment
( Lobert, Graichen, & Morris, 2013 ). The upregulation of miR-200c is associated
with a significant decrease in the miR-200c target, ZEB1, an inducer of EMT.
The transition from epithelial-to-mesenchymal phenotype indicates that cells have
reduced anchoring proteins, such as E-cadherin and Claudin 1. These cancer cells
thus have the capacity to invade tissues and metastasize. Thus, measurements of
ZEB1 inhibition provided evidence for increased amounts and activity of miR-200c.
5.1.3.2 Immunoprecipitation of RISC
Immunoprecipitation of micro-RNAs associated with RISC using Argonaute 2 anti-
bodies is another way to assess the micro-RNA activity. We have used the method to
determine whether drug-induced changes of micro-RNA targets correlate with
changes in micro-RNA activity. An increased amount of a specific micro-RNA as-
sociated with RISC suggests that the activity is increased. In addition, it is expected
that increased amounts of target mRNA would also be found associated with RISC if
the micro-RNA has increased activity. To obtain sufficient amounts of cell lysate, we
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