MicroRNA Detection Methods for Mammalian Cell Lines and Their Applications in Therapeutic Protein Production - MicroRNAs as Tools in Biopharmaceutical Production - page 100

cells, regardless of the model organism (human, mouse or rat) used in the original de-
velopment work. With the recent publication of the Whole Genome Sequencing Data
for a CHO K1 cell line (Xu et al. 2011 ), researchers can expect a rapid increase in
validated miRNA reserach tools for CHO cells and studies for miRNA expression in
biopharmaceutical applications. This review aims to introduce the commonly used
miRNA detection methods in mammalian cells to biopharmaceutical researchers,
in order to aid discovery efforts and eventually tame these “tiny targets” for cell
engineering (Barron et al. 2011b ).
There are some unique challenges for miRNA detection (Git et al. 2010 ; Ham-
mond 2006 ; Li and Ruan 2009 ; Yin et al. 2008 ). MicroRNAs are short—mature
miRNAs are only the size of a typical qRT-PCR primer or probe. They have highly
similar sequences within each miR family therefore detection accuracy often requires
discriminating a single nucleotide. Proper reference gene selection and normalization
for miRNA detection also constitute technical obstacles. Lack of easy purification,
selective enrichment and amplification methods also makes it difficult to detect low
abundant miRNA species. Researchers use different strategies to overcome the tech-
nical obstacles and have successfully developed methods for miRNA detection in
mammalian cells. Among the most commonly practiced are quantitative reverse-
transcriptase PCR (qRT-PCR), microarray and next-generation sequencing (NGS)
based methods (Git et al. 2010 ), all of which have been applied to studies that are
relevant to biopharmaceutical cell culture. The major findings of these studies are
reviewed in the following sections. This review also introduces several novel miRNA
detection methods that have not yet been reported for biopharmaceutical applications
but can be adapted and validated to suit such purposes.

7.2 RNA Isolation for miRNA Expression Analysis

High quality RNA is essential for accurate miRNA detection and profiling. Mi-
croRNA isolation from mammalian cells can be performed using total RNA isolation
methods that preserve small RNAs (Mraz et al. 2009 ). Tri reagent based methods
are a good choice (Rio et al. 2010 ). Users should verify small RNA preservation
when using silica based RNA isolation kits that typically only preserve RNA species
> 200 nt. Some detection methods provide options for small RNA enrichment, which
is of particular importance in next-generation sequencing based methods (Creighton
et al. 2009 ). Storage and handling of miRNA samples is similar to that of total RNA.

7.3 Quantitative RT-PCR

Many researchers consider qRT-PCR the “gold standard” for miRNA detection
(Table 7.1 ). The specificity of miRNA qRT-PCR methods is achieved by incorpo-
rating designs that are unique for miRNAs. The reverse transcription step typically
utilizes a 5 sequence tag in the primer for the first-strand synthesis (Wang 2009 ; Chen
et al. 2005 ; Schmittgen et al. 2008 ). The sequence tag is then used for the reverse