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Review
. 2012 Apr 18;13(5):358-69.
doi: 10.1038/nrg3198.

MicroRNA profiling: approaches and considerations

Colin C Pritchard  1 , Heather H Cheng, Muneesh Tewari
Affiliations
Review

MicroRNA profiling: approaches and considerations

Colin C Pritchard et al. Nat Rev Genet. .

Abstract

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate the expression of thousands of genes in a broad range of organisms in both normal physiological contexts and in disease contexts. miRNA expression profiling is gaining popularity because miRNAs, as key regulators in gene expression networks, can influence many biological processes and also show promise as biomarkers for disease. Technological advances have spawned a multitude of platforms for miRNA profiling, and an understanding of the strengths and pitfalls of different approaches can aid in their effective use. Here, we review the major considerations for carrying out and interpreting results of miRNA-profiling studies.

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Conflict of interest statement

Conflict of Interest Statement: Dr. Tewari is an inventor on patent applications related to circulating microRNA. He has served on the Scientific Advisory Boards of Wafergen, Inc. and Combimatrix, Inc. within the last three years and has had a past research collaboration with scientists at Nanostring, Inc.

Figures

Figure 1
Figure 1. miRNA Biogenesis
Primary microRNAs (pri-mir) are processed by Drosha to pre-miRNA (pre-mir) in the nucleus. Pre-miRNA are exported to the cytoplasm and further processed by Dicer to an miRNA:miRNA* duplex comprised single stranded miRNA derived from the 5′ (5p) and 3′ (3p) regions of the precursor. The duplex is unwound, and one of the strands is loaded into the RISC complex (3p in the figure), while the other passenger strand (miR*) is usually degraded (5p in the figure).
Figure 2
Figure 2. miRNA Profiling Workflow
A) MicroRNAs can be extracted from a variety of specimen types, highlighted here are plasma and serum, cells in culture, fresh tissue/tumor, or fixed tissue/tumors. B) Methods for purifying miRNA populations of interest include size purification by gel electrophoresis, and AGO2 immunoprecipitation (AGO2-IP), with or without UV crosslinking (CLIP). Laser capture microdissection (LCM) can be used to purify material from fresh-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. C) MicroRNA isolation methods are similar to total RNA isolation, and commercially available extraction kits are listed in the light orange boxes. These typically use a chemical extraction combined with a purification step involving binding and eluting from a silica column. Formalin fixed (FF), or formalin fixed, paraffin embedded (FFPE) tissue/tumor require additional deparaffinization and protease treatment, and commercially available kits designed for these sample types are also listed in the light orange boxes. We recommend adding spike-in oligos prior to RNA extraction to enable a quality control check, particularly when using plasma or serum samples. D) A variety of methods can be used to assess miRNA quality after extraction. For most samples, these methods include spectrophotometry, automated capillary electrophoresis with the Bioanalyzer or Experion, and/or determining expression of housekeeping miRNAs. For serum and plasma, which usually have total RNA yields too low to accurately quantify, determining the recovery of spiked-in oligos can be a useful surrogate.
Figure 3
Figure 3. Approaches to miRNA Profiling
(A) qRT-PCR. In TaqMan qRT-PCR the reverse transcription (RT) reactions use stem loop primers specific to the 3′ end of the miRNA for specificity (A, top left). Amplicons are generated using an miRNA-specific forward primer, where DNA polymerase proceeds along template, the TaqMan probe is hydrolyzed so the quencher is freed from fluorescent dye, resulting in light emission (A, top middle). In SYBR green-based qRT-PCR miRNA is polyadenylated at the 3′ end and Oligo d(T) used as an RT-primer (A, bottom left). An miRNA-specific forward primer and oligo d(T) enable PCR amplification with dsDNA-intercalating SYBR green dye as the detector (A, bottom middle). Both TaqMan and SYBR green-based qRT-PCR are available in medium format “array” (A, right). (B) miRNA Microarray. DNA-based capture probes (which may or may not incorporate LNA-modified bases) are used to capture fluorescently-tagged miRNAs, followed by scanning of slides and quantification of fluorescence. Several variations on this approach exist. (C) RNA Sequencing. Current established RNA sequencing platforms begin with reverse transcription of miRNA to a cDNA library. Adaptor ligation then allows the library to either be affixed to a solid phase as in the Illumina platform or to beads for emulsion PCR as in the Roche and ABI platforms. For details of RNA sequencing chemistry see Metzker 2010. (D) Nanostring NCounter: Two target-specific probes are designed for each miRNA of interest: a 3′ capture probe containing biotin to allow adsorbance to solid phase via strepavidin, and a second 5′ reporter probe with an individual color-coded sequence. No amplification or labeling is required with this method.
Figure 4
Figure 4
Choosing an miRNA Profiling Platform.
See this image and copyright information in PMC

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