The study of noncoding RNAs, especially noncoding microRNAs (miRNA), has
gained increasing attention in recent years. MicroRNAs are 18-24 nucleotide
long single stranded RNAs that regulate the expression of target genes by
interacting with complementary sites in the 3’ UTR of the target mRNAs and
inhibiting translation. miRNAs are a conserved group of noncoding RNAs with
very important regulatory roles. miRNAs have been implicated to have
some role in cancer and development.
There are estimated to be over a thousand distinct miRNAs in mammalian cells,
not including the many other small noncoding RNAs.
Methodology
The approach begins with covalent
attachment of an adaptor to the ends of the RNA. This generates
RNA molecules with anchor to one or two ends of the RNA molecules. A reverse
transcription reaction is then initiated with a primer designed to the
adapter. This is followed by PCR amplification with the same primer. This
process is achieved in only a few hours. The amplified cDNA is then cloned
and sequenced. The sequences obtained are first blasted against GenBank.
cDNAs with sequences homologous to entries not derived from rRNA are
then blasted against the
Sanger miRNA database. cDNA sequences not present in the miRNA data bases
are then subjected to further study by both Northern blot and 3’ RACE RT-PCR.
Details of the miRNA-like amplification
method have been submitted for publication.
The following table presents an estimation
of the enrichment factor of MicroRNA after cDNA amplification using SBI’s
MicroRNA Discovery™ Kit:
We estimated the enrichment factor by RT-PCR comparing the number of PCR cycles needed to amplify a
miRNA and a primer pair designed to amplify a segment of the 5S rRNA
transcript. The miRNA selected for this study was miR-16 which was found to
be present in the amplified and non-amplified cDNA. The results are shown above. Before amplification, the difference in the number of cycles needed
to amplify to a similar level the 5S rRNA and miR-16 was +9. After amplification the number of
cycles was -1. The difference was about 10 cycles which suggests an
enrichment factor of about 1,000.
Although obviously not quantitative, an enrichment of a known miRNAs was
clearly achieved.
Conventional Method For MicroRNA Discovery (reference)
The conventional method of miRNA discovery is solely directed at cloning
very small RNAs. In the first step, small RNA molecules (18-24nt) are
obtained by size selection of total RNA (gel electrophoresis, band excision,
and purification). In a second step, an adaptor is ligated to the 3’- end of
the RNA. This is followed by size selection. In the third step, a first
strand cDNA is generated by RT. This is again followed by size selection. In
the fourth step, an adaptor is ligated to the 3’-end of the cDNA strand.
Again this is followed by size selection. Finally, the cDNA is amplified by
PCR, cloned and sequenced. This process can take several days to complete,
and requires at least 10µg of starting total RNA.
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