Table 1.
Primers used in this study.
| A: Oligonucleotides Primers for Cloning miR-199a-1 and miR-199a-2 | ||||
| Primer-1 | Complementary primer | |||
| Pre-mir-199a-1 | 5′agcttGCCAACCCAGTGTTCAGACTACCTGTTCAGGAGGCTCTCAATGTGTACAGTAGTCTGCACATTGGTTAGGCg-3′ | 3′aCGGTTGGGTCACAAGTCTGATGGACAAGTCCTCCGAGAGTTACACATGTCATCAGACGTGTAACCAATCCGctta-5′ | ||
| Sequences were downloaded from miRBase for miR-199a-1, MI0000242. The bold underline marks the sequence of miR-199a-5p (MIMAT0000231) and the bold italic marks the sequence of miR-199a-3p (MIMAT0000232). The lowercase underline letters marks the nucleotides added to the primers to generate HindIII and EcoRI sites. | ||||
| B: Primers used to amplify 3′UTRs | ||||
| Gene symbol | NCBI Reference Sequence: | Forward primer | Reverse primer | Legends of PCR product |
| FN1 | NM_002026.3 | GTCTCGAGCAGCCAACCAAGATGCAAA | TTTTCCTTTTGCGGCCGCAGGTGGAGGGAAGAAGGGAA | 727 |
| SMAD4 | NM_005359.5 | GTCTCGAGTGGGGCAAGACTGCAAAC | TTTTCCTTTTGCGGCCGCCTCATTCACAGTAAAATGGACCT | 877 |
| The added nucleotides to generate XhoI site in the 5′ and NotI of the 3′ PCR product are marked by underline (sequences were added as recommended on the NEB website) https://international.neb.com//media/nebus/files/chartimage/cleavage_olignucleotides_old.pdf?rev=c2f94e1cdcd549c5bf8fdb59f7b63f67&hash=0C83C8F3C59132BE6A5B9A6D4050E3A0. | ||||
| RAP2B | NM_002886.3 | CCCGGGAATTCGTTTGTGGCTCTTTGCAGCATGTA | GGCCGCTCTAGGTTTCAAATTCATTGCAAGAGATGGA | 1033 |
| VANGL1 | NG_016548.1 | CCCGGGAATTCGTTTGCAGGTGTGTAGCTCAGCAG | GGCCGCTCTAGGTTTCCAGAAGTGCCGAATCATTT | 1024 |
| AKT3 | NG_029764.1 | CCCGGGAATTCGTTTTCCACCCTCTGAGACTCCAT | GGCCGCTCTAGGTTTCCAGCTGGGGCTATTAAAAA | 1073 |
| TGFBR3 | NG_027757.1 | CCCGGGAATTCGTTTGGGCTGAGATTTCCAGGCTA | GGCCGCTCTAGGTTTTTGGAGTTTGGGGCATTTTA | 1016 |
| GGA3 | NM_138619.3 | CCCGGGAATTCGTTTGAACCAAACTGCTGCTGTGA | GGCCGCTCTAGGTTTAGCTAGAGTGGCTGGGACAA | 1006 |
| DERL1 | NM_024295.5 | CCCGGGAATTCGTTTTTCTTGCACACATGCCTCTC | GGCCGCTCTAGGTTTTTGCCTCAAAGTGTGACAGC | 1022 |
| SYTL2 | NG_029712.1 | CCCGGGAATTCGTTTAATGAGCCCAAATTCCACTG | GGCCGCTCTAGGTTTGCCCACTTAGGGGAGATGAT | 1066 |
| BIRC5 | NG_029069.1 | CCCGGGAATTCGTTTCTGGGAAGCTCTGGTTTCAG | GGCCGCTCTAGGTTTAGCATCGAGCCAAGTCATTT | 1006 |
| YWHAE | NG_009233.1 | CCCGGGAATTCGTTTTTTAGGTTCCTGCCCTGTTG | GGCCGCTCTAGGTTTCTGGAGGACAAGACACACCA | 1000 |
| TFAP2A | NG_016151.1 | CCCGGGAATTCGTTTGAGCAGGGAAGAGGGTCTTT | GGCCGCTCTAGGTTTCACGGCCTGTTCTGTTCTCT | 1002 |
| GRHL3 | NG_009308.2 | CCCGGGAATTCGTTTCCGTACCCCAAAACAATGTC | GGCCGCTCTAGGTTTGTGCCAACATGACCACACTC | 1060 |
| TNS4 | NM_032865.5 | CCCGGGAATTCGTTTCCCCCTTGCAGATGAGTATC | GGCCGCTCTAGGTTTGCCTGTGACCTTGAGAACCT | |
| The underlined sequences in both sets of primers are homologous to the 15 nucleotides up and downstream of the PmeI cut site on psiCHECK-II. | ||||
| C: Primer used to generate mutation in RAP2B miR-199a-3p binding site | ||||
| Forward primer | Reverse primer | |||
| RAP2B | AAAATCAAAGGGGAGTCTGGG | TCTTGTAAACAAAATAGATTTTTTTTCCACAAATATC | ||
| D: primers used for real-time mRNA real-time RT-PCR | ||||
| Gene name | Forward primer | Reverse primer | ||
| DNM2 | TACATGCTGCCTCTGGACAA | CTGCTCCGTGTTGAAGATGG | ||
| DNM3OS | AGCCTTCCAGTTTGTACCCT | AGGCAGTTGTGAGCTTAAGT | ||
| SMAD3 | ACTACATCGGAGGGGAGGTC | TAGCGCTGGTTACAGTTGGG | ||
| SMAD4 | CGCTTTTGTTTGGGTCAACT | CCCAAACATCACCTTCACCT | ||
| SERPINE1 | GGGCCATGGAACAAGGATGA | CGGAACAGCCTGAAGAAGT | ||
| SERPINE2 | GCAGGACCAAGAAGCAGCTCG | CACGGCGTTAGCCACTGTCACAAT | ||
| COL4A1 | GCCCTTCTGCTCCACGAG | CAGTCACATTTGCCACAGCC | ||
| TWIST1 | GAGCTGGACTCCAAGATGGC | TCCATCCTCCAGACCGAGAA | ||
| TWIST2 | GCAAGAAGTCGAGCGAAGAT | GCTCTGCAGCTCCTCGAA | ||
| ID1 | GGCTGTTACTCACGCCTCAA | TGTAGTCGATGACGTGCTGG | ||
| ID2 | AGGAAAAACAGCCTGTCGGA | GAGCTTGGAGTAGCAGTCGT | ||
| VIM | GGGAGAAATTGCAGGAGGAG | ATTCCACTTTGCGTTCAAGG | ||
| GAPDH | CTGACTTCAACAGCGACACC | GGTGGTCCAGGGGTCTTACT | ||
| RPLPO | CAGATCCGCATGTCCCTTCG | GCAGCAGTTTCTCCAGAGCTGG | ||
| E. Primers used for amplification of putative promoters regions of miR-199a-1 and miR-199a-2 after DNA was subjected to Bisulfite conversion | ||||
| Gene name | Forward primer | Reverse primer | ||
| miR-199a-1 | CGGTACCCGGGGATCTGTTATATTTGGAATTGTTTATA | CGACTCTAGAGGATCCAAACCCAACCTAACCAATATACA | ||
| miR-199a-2 | CGGTACCCGGGGATCGTTTGAAGATGAAATGATTGTTTAA | CGACTCTAGAGGATCCTCCCTTACCCAATCTAACCAATATA | ||
| The underlined sequences in both sets of primers are homologous to the 15 nucleotides up and downstream of the BamHI cut site on PUC19. | ||||