Biology Reference
In-Depth Information
spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). Using this
system, mature miRNAs are polyadenylated and tagged at the 5
0
end. The polyade-
nylation allows oligo DT priming for the reverse transcription. Subsequent PCRs
take advantage of the 5
0
tag to isolate specific mature miRNAs using a universal
primer. Other methods for purifying miRNAs can be found in
Redshaw et al. (2013)
.
Measurement of micro-RNA levels using qRT-PCR with synthesized micro-
RNA oligonucleotides for the standard curve yields the micro-RNA copy numbers
(
Redshaw et al., 2013
). The data can be normalized to cell number or microgram total
RNA. Alternatively, we use comparative real-time PCR to measure micro-RNA
levels in cell culture studies where we have multiple experimental conditions to com-
pare. This method requires the use of preferably two or more housekeeping genes to
normalize data. We have found several reliable housekeeping genes for these studies
(e.g.,
RNU6
,
RNU1A
,
SCA17
,
SNORD44
). Housekeeping genes should be evaluated
experimentally for each set of experiments. The housekeeping gene expression
should not vary by more than
0.5
C
t
(threshold cycle) with drug treatment or other
experimental condition relative to the control samples (
Lobert et al., 2010
).
5.1.1.3
PCR arrays
We use micro-RNA PCR arrays, Cancer PathwayFinder (SABiosciences-Qiagen
Corporation, Valencia, CA), to identify changes in micro-RNA levels associated
with paclitaxel treatment. The miScript miRNA PCR arrays are pathway focused
and are available in 96- or 384-well formats. Separate plates for control and exper-
imental samples are prepared. There is a free web-based analysis tool that uses the
DD
C
t
method to compare raw data for the experimental and housekeeping genes and
calculate relative quantification (
http://www.sabiosciences.com/dataanalysis.php
)
.
A TRIzol-based protocol is used to purify total RNA from cells, and first strand
cDNA synthesis is done according to the superarray manufacturer's instructions.
Then template cDNA is used in the qRT-PCR experiments. Further validation of
micro-RNA changes associated with paclitaxel treatment is performed using the
qRT-PCR validation assays (RT
2
miRNA qPCR Assays, SABiosciences-Qiagen
Corporation, Valencia, CA). These kits provide the buffers and primers needed
for quantification using real-time comparative PCR.
5.1.1.4
Next-generation sequencing
Next-generation sequencing (massively parallel sequencing) technology for DNA
and RNA analysis offers both high-speed data collection and analysis capacity pre-
viously unavailable. Several different platforms can be used for micro-RNA data col-
lection and analysis (
Brown, 2013; Zhang, Chiodini, Badr, & Zhang, 2011
). For
example, the Illumina Genome Analyzer can generate 200 giga basepair (Gbp) of
short read data per run with accuracy greater than 99.5% (
Zhang et al., 2011
).
The technique permits comparison of thousands of genes with known micro-RNA
databases or comparison of unknowns with whole genome sequences. For measure-
ment of drug-induced changes in micro-RNAs, total RNA is purified using a TRIzol-
based protocol and processed for analysis. The sample processing and bioinformatics
