The 3′untranslated region (UTR) of CCL2 containing the putative binding site of miR-206 was identified by searching the online miRNA database (18 (link),19 (link)). The CCL2 3′UTR was amplified using PCR, and the PCR products were isolated using agarose gel (2%). The fragments were then purified and inserted into a pGL3-control vector (Promega Coproration, Madison, WI, USA). Mutagenesis was performed for the same site and inserted into the control vector (Promega Corporation) using PCR. The miR-206 primers were added into the reverse transcription system. The miRNA reverse transcription program is consistent with the common mRNA procedures (20 (link)).
Protocol detail
Identification and Validation of miR-206 Binding in CCL2 3'UTR
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Agarose
Binding site
Ccl2
Mirna
Mrna
Mutagenesis
Pgl3
Primers
Promega
Reverse transcription
Untranslated region
Corresponding Organization :
Other organizations : Taian City Central Hospital, Qilu Hospital of Shandong University
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Variable analysis
independent variables
- Presence of miR-206 binding site in the 3' UTR of CCL2
- Mutagenesis of the miR-206 binding site in the 3' UTR of CCL2
- Expression of CCL2 reporter gene
- PGL3-control vector (Promega Corporation, Madison, WI, USA)
- Common mRNA reverse transcription program for miRNA
- Positive control: Wild-type CCL2 3' UTR in pGL3-control vector
- Negative control: Mutated CCL2 3' UTR in pGL3-control vector
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