Plant MicroRNA validation

Identification and validation of plant MicroRNA has been summarized by Meyers et.al., in the article Criteria for Annotation of Plant MicroRNAs. They build upon the previous article by Ambros et. al., which had expression and biogenesis criteria.

The expression criteria were:

1. Hybridization of a "specific" RNA probe to a size selected RNA sample, using a method like Northern blotting.
2. Identification of the "specific" RNA sequence in a size selected cDNA library. It is expected that this sequence matches the genomic sequence of the organism from which they were cloned.Various sequencing technologies have been used to generate size selected cDNA libraries.

 The Biogenesis criteria were:

1. Structure prediction should support a fold-back precursor that contains the "specific" miRNA sequence within one arm of the hair-pin. Morever, this hairpin should have the lowest free energy as per a RNA-folding program like mfold "and must include at least 16 bp involving the first 22 nt of the miRNA and the other arm of the hairpin". Apart from meeting these criteria, the hairpin should also be free of any internal loops or large asymmetric bulges.
2. The "specific" sequence and its predicted precursor fold-back secondary structure should be conserved.
3. Increased accumulation of precursor in organisms with reduced Dicer function. 

Meeting any of these criteria individually is not sufficient to be considered a miRNA as even siRNA's meet the expression criteria and biogenesis criteria are not specific to miRNA's. Hence, both expression and biogenesis criteria are required for proper validation of miRNA as per Ambrose et.al., The more recent (2008) set of guidelines by Meyers et. al., tries to utilize the knowledge gained by studies in the 5 years since the initial criteria by Ambrose et.al in 2003.

The criteria set up by Meyers et. al., are grouped under primary and ancillary criteria with various precautions to be taken. It also has information about assigning miRNA's to families.

Primary criteria include presence of miRNA sequence in cDNA library and its validation by hybridization experiments and adherence to the expected stem-loop structure. Datasets with low coverage are to be treated with caution as they have chance of mistaking a siRNA as a miRNA.

While ancillary criteria can support a miRNA candidate that meets the primary criteria, it is considered neither necessary nor sufficient for a prediction. However, "clear" conservation is generally sufficient to annotate an miRNA, as long as it has satisfied the primary criteria in the organism in which it has an homolog.  Other criteria such as miRNA target prediction, DCL1 dependence, RDR and PolIV, PolV independence are useful for obtaining biological function information but not enough to validate a sequence as miRNA.

Recently, many new automated pipelines have been published for automating the process of miRNA identification, validation, annotation and target prediction.

1. miRTour has a easy to use web interface to upload the EST/contigs but it has a 50Mb limit on the size of the dataset that can be uploaded.
2. PIPmiR (Pipeline for the Identification of Plant miRNAs) provides an executable that can be downloaded and used.It has been used to predict both known and novel miRNAs in Arabidopsis.
3. shortran: a pipeline for small RNA-seq data analysis is also available for download. 
4. miRDeep-P is a version of miRDeep modified for use with plant transcriptomes.
5. sRNA toolkit is a more general solution that identifies not only miRNA but other small RNA's.

The availability of numerous automated pipelines for plant miRNAs can be useful, but at the same time introduce its own set of artifactual problems.