The Application of MicroRNAs in Cancer Diagnostics - RNA Interference from Biology to Therapeutics - page 262

Fig. 13.3 MicroRNA expression profiling methodologies . Microarray analysis is shown as an
example of hybridization-based miRNA expression profiling ( left panel ). TaqMan-based RT-qPCR
is presented as an example of amplification-based miRNA expression analysis ( middle panel ).
Next-generation sequencing (also known as deep sequencing) is a newly developed technology for
sequencing-based analysis of miRNA expression ( right panel )
for microarray-based miRNA detection. Although higher specificity can be obtained
by incorporation of chemical modifications, such as locked nucleic acid (LNA), into
the probes [ 103 ], it is generally recommended to validate microarray-based findings
by an alternative methodology. Early studies generally used rather laborious
Northern blotting [ 104- 106 ], but today RT-qPCR is typically used (see below). ISH
on tissue sections using labeled miRNA-specific probes (usually LNA probes) has
also been used for validation, although this technology may be more suited for
localization studies than differential expression analysis [ 107 ] . A main advantage of
ISH is that it can be used in combination with tissue microarrays (TMAs), thus
allowing the parallel analysis of a particular miRNA in clinical tumor specimens
from hundreds of patients on a single TMA.
Compared to microarrays and other hybridization-based methods, RT-qPCR has
increased dynamic range and can be used for profiling of very limited amounts of
RNA often available from clinical samples [ 108- 110 ] . The short length of mature
miRNAs and sequence similarities between related miRNAs, and with pre- and pri-
miRNAs, are also challenges for RT-qPCR assay. Reverse transcription is performed
either directly by the use of miRNA-specific primers, including linear and stem-
loop primers (Fig. 13.3 , middle panel), or by universal priming after enzymatic
addition of a polyA tail to template miRNAs (not shown) [ 111 ] . When using univer-
sal priming for RT, some studies suggest that it may be necessary to incorporate