MicroRNA Detection Methods for Mammalian Cell Lines and Their Applications in Therapeutic Protein Production - MicroRNAs as Tools in Biopharmaceutical Production - page 103

a control RNA should have similar stability and size as the miRNAs analyzed in
qRT-PCR (Chen et al. 2005 ; Wong et al. 2007 ), which makes small RNAs better
controls than some traditional housekeeping genes. Koh and colleagues used small
nucleolar RNA U6 as the reference gene. Gammel et al. (Gammell et al. 2007 ) used
5 S RNA as the housekeeping gene for their qRT-PCR studies. Several researchers
selected miR-16 as the internal control miR based on its expression stability in
various experimental conditions and tissues (Peltier and Latham 2008 ; Wong et al.
2007 ; Lin et al. 2010 ). A very recent publication by Druz et al. (Druz et al. 2011 ) used
let-7c as the reference gene for qRT based on their microarray data in CHO cells.
Increasing reports describe effective applications of algorithms such as Norm Finder
for evaluating and selection of reference genes for miR qRT in various cell lines and
organisms (Peltier and Latham 2008 ; Wotschofsky et al. 2011 ; Zhu et al. 2011 ; Chen
et al. 2011 ; Brattelid et al. 2011 ; Schaefer et al. 2010 ; Carlsson et al. 2010 ; Latham
2010 ; Mortarino et al. 2010 ). The Vandesompele lab who has published extensively
on qRT-PCR normalization proposed using the mean expression value of all miRNAs
as the normalizer (Mestdagh et al. 2009 ). All of these normalization methods should
be easily applied to CHO cells.

7.4 Microarrays

In the past decade, microarray-based methods have matured and become standard
practice for transcriptional profiling. Continued discovery of new miRNAs has led
to rapid expansion of miRBASE (Griffiths-Jones et al. 2006 , 2008 ): v17 contains
16772 hairpin precursors representing 19724 mature miRNA products in 153 species.
Humans alone are reported to have 1,492 miRNAs. As a result, array-based profiling
becomes increasingly attractive to perform studies in which qRT-PCR does not pro-
vide sufficient throughput. Numerous types of commercially available and custom
made microarray platforms have been developed and updated with the discovery of
miRNAs. The small size of the miRNAs constitutes one of the technical challenges
for probe design. Current oligonucleotide array platforms use similar principles and
workflow of detection as DNA arrays: probe immobilization, target labeling and
hybridization. For probe design, the oligonucleotide arrays incorporate modified nu-
cleic acids such as Locked Nucleic Acids (LNAs) (Castoldi et al. 2007 ) or simply
increase probe sizes in order to normalize melting temperature, and achieve high
affinity and single-nucleotide discrimination (Li and Ruan 2009 ; Yin et al. 2008 ).
An extended hairpin design in addition to the hybridization sequence has been suc-
cessfully applied to increase specificity and stabilize probe-target interaction (Wang
et al. 2007 ). Bort and colleagues used a custom LNA based microarray platform
and reported successful differential expression analysis of miRNA expression in lag,
exponential, stationary and decay phases of CHO K1 batch cultures (Bort et al. 2011 ).
While dual- or single-fluorescent probe labeling and hybridization on glass slide
array platforms remains the most common, many researches developed their own
custom oligo DNA arrays for miRNA profiling with various surface chemistry,