MicroRNA Detection Methods for Mammalian Cell Lines and Their Applications in Therapeutic Protein Production - MicroRNAs as Tools in Biopharmaceutical Production - page 102

qPCR primer binding, in order to increase specificity of the qRT-PCR and lengthen
the amplicon for detection. For example, Chen and colleagues use a stem-loop de-
sign for the miR-specific RT primers that allow detection and to distinguish between
precursors and mature miRNAs (Chen et al. 2005 ; Schmittgen et al. 2008 ). Another
approach is to include a 5 universal tag in the oligo dT RT primer after adding a
polyA tail to the mature miRNAs (Jacobsen et al. 2011 ), and the 5 -universal tag is
used as a miR-specific reverse qPCR primer binding site.
It is not surprising that researchers selected qRT-PCR, one of the most well-
established molecular biology methods, in early miRNA studies before CHO genome
sequences were publicly available. Gammell and colleagues investigated the miRNA
expression changes in CHO K1 cells induced by cold shock and published the first
paper examining miRNA expression in CHO (Gammell et al. 2007 ). They used qRT-
PCR as their method for differential expression analysis. The majority of subsequent
studies published in CHO miRNA all used qRT-PCR for differential expression
analyses, either as the primary method or as the confirmatory method following
microarray experiments (Barron et al. 2011a ; Druz et al. 2011 ; Lin et al. 2010 ).
While miR-specific RT provides great specificity, it limits the throughput and
increases cost for miRNA qRT-PCR. Simply pooling miR-specific primers for uni-
versal RT may lead to interference between miRs and decrease detection sensitivity
and specificity for the lower abundant miRs. The polyA addition followed by oligo
dT priming method reviewed previously can be used to achieve universal RT reac-
tion; specificity is achieved by strategies in qPCR such as incorporating LNAs in
the primers (http://www.exiqon.com/microrna-real-time-pcr). This approach, natu-
rally, can be used to design qRT panels. Another common strategy for universal RT
is to design panels with limited number (up to 381) of miR qRT assays, then per-
form RT of these miRNAs after experimental validation of specificity and sensitivity
(https://products.appliedbiosystems.com). A novel bioinformatics method partitions
miRNA sequences and subsequently balances RT annealing temperature homogene-
ity, primer dimer formation and sequence cross-reactivity to achieve simultaneous
qRT-PCR of 96 cancer-related miRNAs (Wang 2009 ).
Utilizing some of the techniques to increase throughput, researchers now routinely
take “quantitative PCR array” approaches that retain the advantages of qRT-PCR,
but utilize high-throughput 96- or 384-well multiplexed qRT assays that focus on
various pathways (Chen et al. 2009 ). Among the most recently developed methods is
Taqman Low Density Array (TLDA) (Zhang et al. 2010 ; Gokhale et al. 2010 ; Wang
et al. 2011 ) that uses microfluidics cards to format qRT-PCR reactions with very
small reaction volumes to increase throughput of qRT.
Mechanistic studies on miRNA biogenesis call for detection of miR precursors
(pri-miRs and pre-miRs) that are longer in length (Lee et al. 2002 ). The detection
of the precursor hairpin requires thermo stable RT, since denaturing the hairpin is
essential for first-strand synthesis. Although the stable hairpin exists in both pre-
cursors, some researchers choose to amplify the larger pri-miRNA precursor due to
increased sensitivity and linearity that they observed (Schmittgen et al. 2008 ).
Last but certainly not least, normalization has always been a point of debate since
the discovery of miRNA, even more so in the less characterized CHO cells. Ideally