ABSTRACT
During the last decade, quantitative real-time PCR (qPCR) has reached its full potential as an essential tool for use in the quantitative analysis of nucleic acids. qPCR is the method of choice for validating results obtained from whole-genome screening (e.g., with microarray or Next Generation Sequencing) and in the screening for a clinically signicant, pre-dened subset of genes to be evaluated in patients as predictive1-3 biomarkers. The intrinsic nature of microRNAs (miRNAs) and their growing importance as diagnostic markers has required the development of dedicated technologies to perform detailed and reproducible analysis, starting with RNA extracted from various sources and with a different range of methods. Currently, biomedical researchers aim to analyze miRNA expression routinely in archival collections of formalin-xed paraf-n embedded (FFPE) material,4,5 in RNA extracted from laser-capture microdissected (LCM) samples6,7 as well as from single-cells.8,9 Additionally, novel approaches have been developed to analyze serum and plasma circulating nucleic acids.10-14 With the increased demand to achieve complete expression-proles of miRNAs from a minute amount of material and the technical
22.1 Overview ............................................................................................................................307 22.2 Introduction ........................................................................................................................308 22.3 What Are miRNAs? ...........................................................................................................308 22.4 Why Is miRNA Quantication of Interest? .......................................................................309 22.5 Challenges to miRNA Expression Proling ......................................................................309 22.6 RNA Isolation and Quality Control ................................................................................... 310 22.7 Alternative Approaches to cDNA Synthesis ...................................................................... 311
22.7.1 cDNA Synthesis Using miRNA-Specic RT Primers ........................................... 311 22.7.2 cDNA Synthesis by Tailing RNAs ......................................................................... 313
22.8 Noncommercial Alternatives for the Expression Proling of miRNAs: miQPCR Assay .....314 22.8.1 Challenge of miRNA-Specic Primer Design ....................................................... 314
22.9 How to Discriminate among Mature, Pre-, and Pri-miRNAs ........................................... 316 22.10 Detection of Amplied Products ........................................................................................ 316 22.11 Assessment of Specicity and Sensitivity, Data Analysis, and Normalization ................. 318 22.12 What Is New on the Horizon? Expression Proling of Circulating miRNAs ................... 318 22.13 Conclusion .......................................................................................................................... 319 References ...................................................................................................................................... 319
challenges associated with each methodology, miRNA proling is slowly reaching the limits of established qPCR techniques.
