36
| 2 | 2011
Klinisk Biokemi i Norden
(
Fortsat fra side 35)
miRNA diversity
miRNA coding genes are often localized in intergenic
regions of the genome, where they are organized
as separate genes, in tandem, head to tail, or in line
configuration. These miRNA genes are preferentially
regulated by their own independent promoters (13).
Alternatively, miRNA genes can be located within other
transcriptional units, usually in intronic regions but also
in exons, where the miRNA expression is regulated in
parallel to their host genes. Multiple gene loci may gen-
erate the same mature miRNA, but these genes are most
often under different regulatory control. It is estimated
that as much as 5 % of the human genome may code
for different miRNAs. The presence of DNA sequence
variations in the miRNA gene, like Single Nucleotide
Polymorphisms (SNPs), small base-deletions or inserts,
as well as epigenetic factors, can contribute to a switch
in the regulatory repertoire. Similarly, genetic variations
in miRNAs may either impair or augment a miRNA-
binding, thereby affect miRNA efficiency.
The Sanger miRBase
) is
one of the most comprehensive miRNA databases
developed, bringing together the miRNA gene nomen-
clature and sequences with automated pipeline for
prediction of miRNA target genes in several animal
genomes. As of today 1048 different human miRNAs
have been identified (miRBase release 16.0). Different
miRNAs are identified through sequential numerical
nomenclature in the database, whereas species are
assigned by a three or four letter prefix. By this nomen-
clature, human miRNAs (
homo sapiens
)
are designated
hsa-miR followed by a non-continuous numbering
from 1 up to 4330, for example hsa-miR-21. Mature
miRNAs are designated miRs, while precursor miR-
NAs are labelled mirs. miRNAs differing in only one
or two sequence positions are given lettered suffixes,
for example hsa-miR-30a and hsa-miR-30b. Identical,
mature miRNAs with distinct hairpin loci in precur-
sors have numbered suffixes, e.g hsa-miR-128-1 and
hsa-miR-128-2.
miRNA technology and analysis
To explore the biology and function of these powerful
mediators, it has been important to develop tools for
miRNA isolation and amplification and to identify the
expression of miRNAs in different cells and tissues (14).
Although the small sized miRNAs create challenges for
their detection and quantification, recent adaptations
of existing technologies have emerged by the use of
miRNA-specific RT-qPCR, microarrays, high through-
put- and whole genome sequencing analysis.
With the short miRNA strands, a potential for cross-
hybridization of miRNAs with high homology can
represent a problem during microarray analysis, espe-
cially when distinguishing between miRNAs that differ
by only one or two nucleotides. The use of synthetic
nucleotides locked in a favourable Watson-Crick posi-
tion (LNA - locked nuclear acids) is one of the miRNA
techniques with improved sensitivity and specificity.
miRNA expression
Quantification of miRNAs in tissues during physiologi-
cal and different pathological conditions has been an
important first step to decipher the broad distribution
and level of miRNAs in human subjects. Comparing
matched samples from healthy individuals with dis-
(1)
Can we measure the marker?
-
Pre-analytical stability excellent
-
Time consuming extraction process that require
expert knowledge
-
Current analytical platforms may be suboptimal
-
Lack of established criteria for normalization
Status:
Several challenges remaining regarding ana-
lytical aspects
(2)
Does the marker add new information?
-
Independent association with diagnosis and/or
prognosis in multivariate regression models
-
Improves the AUC/C statistics of established risk
models
-
Improves fit of established risk models
Status:
Currently not been examined for miRNA
biomarkers
(3)
Will the marker help improve patient
management?
-
Provide information for titration of drug doses
-
Identify patients requiring urgent invasive pro-
cedures
-
Help guide follow-up
Status:
Information on kinetics and pathophysio
logy driving the candidate miR markers required
Table. Status of microRNA biomarkers according to pre-
specified criteria for assessment of novel cardiac biomarkers
(
modified from ref. 19)
