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MicroRNA Discovery
MicroRNA Cloning Overview
MicroRNAs (miRNAs) are an important class of small RNAs that
regulate protein translation (see Getting Started with MicroRNA
Investigation). To date, 230 mammalian miRNAs have
been identified and cataloged in the Sanger miRNA Registry.
Some scientists believe that the number of miRNAs could exceed
1000 [1]. While cloning and sequencing were used to identify
most of the known mammalian miRNAs, increasing effort is being
put into bioinformatic prediction of new miRNA sequences. A combination
of computational biology tools and molecular cloning methods
will likely be used for characterizing additional miRNAs.
Isolating the mature miRNA fraction is
the first critical step in cloning these small regulatory molecules.
Ambion scientists have developed two sequential systems that
are optimized for isolating and enriching RNA preparations
that contain 17–24
nucleotide, mature miRNAs. Here we describe the process by which
miRNAs can be cloned in an effort to identify new members of
this small RNA family.
1. Isolate total RNA using a method
that will capture small RNAs
•mirVana miRNA Isolation
Kit includes small RNAs in the total RNA preparation
and can subsequently be used to enrich for RNA <200 nt
2. Separate mature
miRNAs from precursor molecules
•flashPAGE
Fractionator System will quickly and easily
purify mature miRNA
3. Ligate 3' donor
oligonucleotide to RNA with T4 RNA ligase
•The donor
oligonucleotide may contain deoxynucleotides or both ribonucleotides and deoxynucleotides
•This donor
oligonucleotide should contain a 3' end blocking group (e.g.,
amino group, adenylation) to prevent self ligation and ligation
to the RNA 5' ends
•The donor oligonucleotide should be 5' phosphorylated
•The donor oligonucleotide should contain a restriction enzyme
site that is compatible with your cloning vector
•Example:
5'-pCTGTAGGCACCATCAAx-3' [2] (p=phosphorylation,
bold type=restriction site, x=blocking group)
4. Gel purify RNA
•Remove excess
3' oligonucleotides, enzymes, and salts
5. Ligate 5' acceptor oligonucleotide to RNA with
T4 RNA ligase
•This acceptor
oligonucleotide will contain both ribonucleotides and deoxynucleotides
•The acceptor oligonucleotide should contain the same restriction
enzyme site that is in the 3' donor oligonucleotide
•Example: 5'-ATCGTaggcacctgaaa-3' [2]
(upper case=DNA, lower case= RNA, bold type=restriction site)
6. Gel purify RNA
•Remove excess 5' oligonucleotides, enzymes, and salts
7. Use RT-PCR to amplify RNA
• RT
and PCR primers are based on acceptor and donor oligonucleotide
sequences used above
8. Digest with restriction enzyme whose target
site was incorpo-
rated into donor and acceptor oligonucleotides
9. Concatamerize PCR products with T4 DNA ligase
and fill in the
ends of the PCR products
10. Isolate ligation products (400–600
nt range) by gel purification
•Resulting clones contain 4–6 inserts and
provide longer templates for efficient sequencing analysis
11. Clone into your
cloning vector of choice
12. Screen for vectors containing inserts by PCR
13. Purify vector/insert and sequence inserts
Additional details have been described
by leading miRNA researchers [2–5].
Summary
This is one method that has been used to uncover new miRNAs.
When moving into validation and functional studies, we recommend
using Pre-miR miRNA Precursor Molecules and Anti-miR miRNA
Inhibitors to alter miRNA activity in a cell system. See www.ambion.com/miRNA for more information.
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