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miRNA profiling
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The Microarray Centre currently offers miRNA profiling services on the Affymetrix, Agilent and NanoString platforms.

MicroRNA (miRNA) is regulatory RNA found in plants, nematodes, plants, and animals. miRNA has been implicated in a number of cellular processes (for example, developmental timing, proliferation, programmed cell death, fat metabolism, and cancer), and, in mammals, is thought to act as post-transcriptional modulator of gene expression.

miRNA occur in three different forms: long pri-miRNA, hairpin pre-miRNA, and short mature miRNA. In the nucleus, pri-miRNA is cleaved by the nuclease Drosha to form pre-miRNA. Pre-miRNA is 70-125 nucleotides long and form hairpin structures with an overhang of 2 nucleotides at the 3’ end. Pre-miRNA is exported into the cytoplasm by RanGTP and exportin5 proteins. In the cytoplasm, pre-miRNA is processed by the Dicer ribonuclease to form the mature miRNA. Mature miRNA are 19-23 nucleotides long.

miRBase is a public miRNA database created by the Wellcome Trust Sanger Institute. miRBase incorporates the database and gene naming roles, and contains 3 main sections including miRBase Sequences (all published miRNA sequences, genomic locations and associated annotation), miRBase Targets (a database of predicted miRNA target genes), and miRBase Registry (confidential service assigning official names for novel miRNA genes prior to publication of their discovery).

As with protein-coding mRNA, a key to understanding the role of miRNA is to determine when and where they are expressed. The goal of profiling miRNA expression is to discover the specific mRNA molecules regulated by each miRNA molecule.

Microarrays are a good method for high-throughput expression profiling, however, one of the challenges of profiling miRNA expression is that the small size of the miRNA entities leaves little room for labelling or designing specific probes. Another challenge is the fact that miRNA exists in three forms (pri-miRNA, hairpin pre-miRNA and short mature miRNA). Expression profiling of short mature miRNA will require that signal from hairpin pre-miRNA and pri-miRNA can be eliminated. In addition, microarrays made with standard DNA probes are often unable to distinguish single nucleotides. Despite these challenges, researchers have successfully used microarrays to profile miRNA expression.

Quick questions:
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What are miRNAs?

MicroRNA (miRNA) is regulatory RNA found in plants, nematodes, plants, and animals. miRNA has been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death, fat metabolism, viral infections, and cancer. In mammals, microRNA is thought to act as post-transcriptional modulator of gene expression.

What is “mature” miRNA?

miRNA occur in three different forms: long pri-miRNA, hairpin pre-miRNA, and short mature miRNA. In the nucleus, pri-miRNA is cleaved by the nuclease Drosha to form pre-miRNA. Pre-miRNA is 70-125 nucleotides long and form hairpin structures with an overhang of 2 nucleotides at the 3’ end. Pre-miRNA is exported into the cytoplasm by RanGTP and exportin5 proteins. In the cytoplasm, pre-miRNA is processed by the Dicer ribonuclease to form the mature miRNA. Mature miRNA are 19-23 nucleotides long.

Why profile miRNA expression?

As with protein-coding mRNA, a key to understanding the role of miRNA is to determine when and where they are expressed. The goal of profiling miRNA expression is to discover the specific mRNA molecules regulated by each miRNA molecule.

What are the challenges of using microarrays to profile miRNAs?

Microarrays are a good method for high-throughput expression profiling, however, one of the challenges of profiling miRNA expression is that the small size of the miRNA entities leaves little room for labelling or designing specific probes. Another challenge is the fact that miRNA exists in three forms (pri-miRNA, hairpin pre-miRNA and short mature miRNA). Expression profiling of short mature miRNA will require that signal from hairpin pre-miRNA and pri-miRNA can be eliminated. In addition, microarrays made with standard DNA probes are often unable to distinguish single nucleotides. Despite these challenges, researchers have successfully used microarrays to profile miRNA expression.

What are the features on Agilent’s miRNA microarray?

Agilent has 3 types of miRNA microarrays: human, mouse and rat. Each slide contains 8 identical arrays (8x15k) and each array contains over 15,000 60-mer oligonucleotides probes generated from recent versions of Sanger miRBase. Probes are representative of mature miRNA species and allow for increased sensitivity due to their hairpin design.

How much RNA is required?

100 ng at a concentration of 50 ng/ul is required. This allows us ample RNA for the labelling reaction and for quality check of the RNA using the Agilent Bioanalyzer and the Nanodrop Spectrophotometer with some remaining in case a repeat is necessary.

Should I provide miRNA-enriched RNA, miRNA only, or total RNA?

The Agilent miRNA labelling protocol recommends total RNA; however, you must be certain to use an extraction ethod that does not eliminate the small RNA components. At this time miRNA enriched RNA and fractionated miRNA are not recommended.

Which extraction method is recommended?

Agilent recommends using Qiagen’s miRNeasy Mini Kit, Applied Biosystem miRVanaTM RNA Isolation Kit, or Invitrogen’s TRIZOL reagent. For the isolation kits, it is recommended that you follow the total RNA extraction protocols. With TRIZOL® reagent care must be taken that all residual TRIZOL is removed from the extraction as any remaining will negatively effect the labelling reaction. Additional chloroform extractions can be performed in order to completely remove TRIZOL® from the extraction (this will slightly decrease the overall yield of the RNA).

What are the quality requirements on RNA to be used for miRNA labelling?

Total RNA must have a 260/230 UV-Absorbance ratio greater than 1.8 and a RIN greater than 7.5. We will check sample quality using the Agilent Bioanalzyer total RNA Nano assay to ensure RNA is not degraded which would result in a RIN less than 7.5). We will also check 260/230 ratio on the Nanodrop. Customers will have the option of continuing with their experiments if their samples do not pass QC, however, we will not guarantee the results that will be obtained.

During labelling, are microRNAs selectively labelled?

Agilent’s miRNA Labelling Reagent and Hybridization kit use a novel Cyanine 3-nucleotide (cyanine 3-Cytidine bisphosphate (pCp)) which selectively labels and hybridises mature miRNAs. The Agilent platform is a 1-colour assay.

If I opt for the data analysis service, what data will I receive?

The data analysis service includes data quantified with Feature Extraction software and loaded into GeneSpring for statistical analysis.

What are the features on Illumina's miRNA BeadChip?

Illumina offers human and mouse miRNA microarrays. Each miRNA slide has a "12-up" design. The 50-mer oligonucleotides probes are generated from recent versions of Sanger miRBase.

What are the quality requirements on RNA to be used for miRNA labelling?

Total RNA must have a 260/230 UV-Absorbance ratio greater than 1.8 and a RIN greater than 7.5. We will check sample quality using the Agilent Bioanalzyer total RNA Nano assay to ensure RNA is not degraded which would result in a RIN less than 7.5). We will also check 260/230 ratio on the Nanodrop. Customers will have the option of continuing with their experiments if their samples do not pass QC, however, we will not guarantee the results that will be obtained.