MicroRNA Detection Methods for Mammalian Cell Lines and Their Applications in Therapeutic Protein Production - MicroRNAs as Tools in Biopharmaceutical Production - page 104

printing technologies, probe design and labeling techniques (Liu et al. 2008 ;

Git et al. 2010 ).
Git and colleagues comprehensively evaluated six commercially available array
platforms for miRNA profiling, and concluded that Agilent, Illumina and Exiqon
microarrays demonstrated least interarray variation, while Exiqon array stood out
for good signal to noise ratio. Agilent, Illumina and Ambion arrays demonstrated the
best correlation with qRT-PCR in this study. All of the cited array platforms may be
suitable for CHO miRNA discovery work, either by using custom-designed probes
against sequenced CHO miRNAs, or by using human/mouse/rat arrays for detecting
highly conserved miRNAs in CHO. Although the Git study was not performed in
CHO, it can be used as a good reference for selecting array platform for CHO based
on the authors' comprehensiveness.
Nevertheless, the technical challenges for miRNA arrays remain. Besides similar
inherent difficulties with qRT-PCR, RNA amplification to increase signal intensity
is not yet proven to be applicable to miR arrays. Contrary to most linear amplifica-
tion labeling protocols for DNA arrays (Ginsberg 2005 ), only miRNA precursors,
not mature miRs, can be amplified through indirect labeling (Liu et al. 2004 ). It is
critical to have a one to one ratio between the precursor and mature miRNA for accu-
rate detection after amplification. However, miRNA precursors and mature miRNAs
often deviate from one to one ratio due to post-transcriptional regulation in miRNA
biogenesis (Lee et al. 2008 ; Thomson et al. 2006 ). Hence potential precursor contam-
ination (Yin et al. 2008 ) may lead to amplification bias and skewed results. Without
effective signal amplification, it can be difficult to achieve an acceptable signal to
noise ratio and low background solely by optimization of washing temperature and
duration, which remains the case for miRNA arrays.
A novel hybridization based technology, microfluidic Paraflo ® chips that can

overcome some of the signal to noise challenges discussed above, has come into play
as an alternative to glass slide microarrays. The platform enables massive parallel
synthesis of probes in picoliter-scale reaction chambers. The miRNA is dual-color la-
beled, and hybridization is conducted in these reaction chambers. The detection sen-
sitivity and accuracy of this technology has attracted researchers to perform miRNA
profiling in various species (Vorwerk et al. 2008 ; Zhu et al. 2007 ; Guo et al. 2009 ;
Ding et al. 2009 ; Zhang et al. 2008 ). Druz and colleagues successfully employed cus-
tom

μ

Paraflo ® chips that included 714 mouse and rat miRNA probes for the discovery
of apoptosis regulatory miRNAs, and identified the pro-apototic miRNA, mmu-
miR-466 h in CHO cells (Druz et al. 2011 ). After this pioneering work,

μ

Paraflo ®
chips may become a common tool for CHO miRNA profiling as the field grows.

μ

7.5 Other Hybridization Based Methods

μ

Bead-based hybridization for miRNA detection utilizes 5 m polystyrene beads

coated with oligonucleotide probes to specifically capture biotinylated miRNA. The
hybridization is carried out in the bead suspension, in contrast to the glass array
hybridization methods discussed in the previous section. The BeadArray method