Download G ENNOVATIONS Genome-wide miRNA Analyses Genomics Core Newsletter

GENNOVATIONS
Genomics Core Newsletter
Volume 1, Issue 7
Genome-wide miRNA Analyses
MicroRNAs (miRNAs) are an evolutionarily conserved class of small 19-22 nucleotide RNAs that
are present in the cell and perform a variety of functions including the regulation of gene expression.
Traditionally, miRNAs are known to regulate gene expression by binding to the 3’ untranslated region of
an mRNA and either targeting that mRNA for degradation or blocking translation to protein. More
recently, additional functions of miRNAs have been elucidated including their use as biomarkers of
disease pathology, and their putative role in intercellular signaling.
Analysis of miRNAs from cells, tissues and body fluids using a next generation sequencing
platform is termed miRNA-seq. MiRNA-seq is useful for identifying the miRNAs (both known and
unknown) present in any given sample and for determining how miRNAs change in response to disease
pathology. For example, the miRNA profile determined from a colon cancer tissue sample is very
different than that of normal colonic epithelial tissue1. Furthermore, miRNAs have been found in the
blood and the profile of these circulating miRNAs changes when the patient develops cancer 2 or is
infected with a virus3, suggesting that
these molecules may be useful
biomarkers.
How does miRNA-seq work?
The initial starting material is either total
RNA or purified small RNA fragments.
If choosing to purify small RNA
fragments from your sample, be sure to
use a kit that is specifically designed for
this purpose, such as the mirVana kit by
Life Technologies or the miRNeasy kit
from Qiagen. Many of the standard kits
commonly used to isolate total RNA are
designed to isolate larger mRNA species
and wash away the smaller miRNAs.
Since most mature miRNAs have a 5'phosphate and a 3'-hydroxyl group as a
result of the cellular pathway used to create them, 3' and 5’ adapter molecules were specifically designed
to target microRNAs and other small RNAs. These miRNA-specific adapters are ligated to the ends of the
miRNA molecule and an RT reaction is used to create cDNA. The cDNA template is then PCR amplified
using a common primer (homologous to the 5’ adapter sequence) and a primer containing one of 48 index
sequences (homologous to the 3’ adapter sequence, plus index sequence). The index sequence, or barcode,
is a known nucleotide sequence that allows many samples to be pooled for the sequencing analysis. Once
the miRNAs have been amplified by PCR, they are gel purified and used as the template for next
generation sequencing (See Gennovations, Volume 1, Issue 1) to identify all of the miRNAs present in the
sample.
References and for more information:
1. Reid, J.F., et al. “miRNA profiling in colorectal cancer highlights miR-1 involvement in METdependent proliferation.” Mol Cancer Res 2012; 10:504-515, doi: 10.1158/1541-7786.MCR-11-0342.
2. Hofsli, E., et al. “Identification of serum miRNA profiles in colon cancer.” British J Cancer 2013;
108:1712-1719, doi: 10.1038/bjc.2013.121.
3. Zhu, Z., et al. “Comprehensive characterization of serum microRNA profile in response to the
emerging Avian Influenza A (H7N9) virus infection in humans.” Viruses 2014; 6:1525-1539, doi:
10.3390/v6041525
Judy Crabtree, Ph.D.
Director
CSRB 748D
[email protected]
504-568-2963
Chris Taylor, Ph.D.
Bioinformatics
CSRB 7605
[email protected]
504-568-2215