Biology Reference
In-Depth Information
Chapter 10
Quantitative RT-PCR Specifi c for Precursor
and Mature miRNAs
Hannah Zöllner , Stephan A. Hahn , and Abdelouahid Maghnouj
Abstract
Quantifi cation of microRNAs (miRNAs) in cells or primary tissues is one of the most important steps in
elucidating their biological functions. However, miRNAs are challenging molecules for PCR amplifi cation
due to the stable hairpin of the precursor form and the small size of the mature miRNA, which is roughly
the same length as the primers used in standard PCR. To date, different assays were introduced for the
specifi c and sensitive quantifi cation of the mature form of miRNAs. In this chapter we describe the extrac-
tion of RNA from microdissected tissue and the quantifi cation of miRNAs using two different methods
(stem-loop qRT-PCR and polyT adaptor qRT-PCR).
Key words
microRNA, Stem-loop PCR, TaqMan
®
, polyT adaptor PCR, qRT-PCR
1
Introduction
Accurate information of spatiotemporal expression patterns of
miRNAs is a crucial step for understanding their functions, requir-
ing quantitative detection technologies specifi c for the active
(mature) miRNA rather than the inactive pre-miRNAs. Specifi c
detection of the mature miRNA molecules by conventional RNA
techniques is challenging, mainly due to their small size, end poly-
morphisms, heterogeneous GC content, and sequence homology.
In order to reach this goal, adaptions of the standard quantitative
reverse transcription PCR (qRT-PCR) have been established for
the detection of miRNAs and are now widely used. Standard qRT-
PCR primer design is incompatible with miRNA detection because
the short size of the target molecule prohibits the accommodation
of both forward and reverse PCR primers. Two technologies were
introduced, the so-called stem-loop qRT-PCR and polyT adaptor
qRT-PCR to overcome this limitation [
1
,
2
].
