MicroRNA Detection Methods for Mammalian Cell Lines and Their Applications in Therapeutic Protein Production - MicroRNAs as Tools in Biopharmaceutical Production - page 105

for miRNA detection is adapted from the cDNA-mediated annealing, selection, ex-
tension and ligation assay (DASL ® assay) (Fan et al. 2004 ). The labeled beads are
randomly assembled and distributed for fluorescent analysis on a 96-well plate Ar-
ray Matrix (Chen et al. 2008 ). Alternatively, the captured miRNA is detected by
streptavidin-phycoerythrin followed by flow cytometric analysis (Lu et al. 2005 ).
The beads can be labelled with up to 100 colors, which allows for numerous com-
binations of miRNAs (Biscontin et al. 2010 ; Siegrist et al. 2009 ; Wang et al. 2011 ).
Bead arrays are considered by some to have higher sensitivity, accuracy and flexi-
bility but lower cost than glass arrays, but have not yet been reported to be used in
CHO miRNA studies.
Northern blotting, a well-established RNA analysis method, has been successfully
applied to both mature and precursor miRNA detection. Researchers incorporated
LNA in their probe design and optimized washing conditions to increase sensitivity
and they also successfully increased resolution by altering cross-linking or elec-
trophoresis procedures (Koscianska et al. 2011 ; Kim et al. 2010 , Pall and Hamilton
2008 ; Varallyay et al. 2008 ; Valoczi et al. 2004 ). Lusi and colleagues developed
a novel label-free electrochemical detection method that incorporates an inosine-
modified guanine free capture probe. They reported detection sensitivity to be 0.1
pmol (Lusi et al. 2009 ). These methods can also be applied to CHO miRNA research.
As mechanistic studies in CHO advance, the need for in situ detection of miRNAs
will increase. In situ detection for miRNAs can elucidate questions such as differen-
tial miRNA expression in sub-cellular compartments or co-localization of miRNAs
and their putative target mRNAs. Probe design and labeling again is the key to
the success of this technique. Early approaches include using hapten-conjugated or
radioactively labeled RNA oligonucleotide probes (Thompson et al. 2007 ) and LNA-
enhanced probes (Kloosterman et al. 2006 ; Nelson et al. 2006 ; Pena et al. 2009 ).
A recent publication reported single-cell miRNA quantitative detection and imag-
ing using LNA probes (Lu and Tsourkas 2009 ), which can be useful for evaluating
miRNA expression in a cell-based fashion in CHO cell populations, or for miR target
co-localization studies.

7.6 Next-Generation Sequencing (NGS) for miRNA Profiling

In the past three to four years, NGS methods have flourished in genomics and
transcriptomics research. Sample preparation protocols have been simplified, and
costs greatly reduced, compared with when NGS was in its infancy. These methods
allow for absolute quantification of target sequences with greater dynamic range
and remarkably higher throughput per experiment than microarrays (Schuster 2008 ;
Buermans et al. 2010 ). Some of the drawbacks of hybridization based technolo-
gies discussed earlier can be avoided using NGS. The read lengths (50-100 bp)
for the two commonly used NGS platforms for small RNA sequencing (Illumina
Genome Analyzer by Illumina and SOLiD by Applied Biosystems), albeit much
shorter than Sanger sequencing, are well-suited for small RNAs and their precur-
sors (Creighton et al. 2009 ). The procedures for miRNA profiling using NGS start