Table 3.
Sequences of oligos used for amplification in qRT-PCR
| Oligo Name | Oligo Sequence | mRNA/cDNA section amplified (bp from ATG) | Total length of mRNA (bp) |
|---|---|---|---|
| Igl 5' F | GCTGTTCCACATTGTGCATCAGTTTCAAATG | 85–450 (Igl1), 85–459 (Igl2) | 3306 (Igl1), 3318 (Igl2) |
| Igl 5' R | TTCTGCATGATCTTCTGTAGTTGCATTATCACATAAC | ||
| Igl 3' F | TGAAGGCACTTCTACAGAAGATAATAAAAT | 2967–3166 (Igl1), 2979–3178 (Igl2) | |
| Igl 3' R | TATGTCTTGAACATGGAATACATGATC | ||
| Igl1 F | TCTTGTAATAAGTTCCCGGAGCA | 634–841 (Igl1) | |
| Igl1 R | CATCAGAAACAGTACATCTTTTATTACATG | ||
| Igl2 F | GTACTAAATACCCAGATCATTGTTCAAA | 643–841 (Igl2) | |
| Igl2 R | CATCAGAAACAGTACATCTTTTATTACATG | ||
| URE3-BP 5' F | CCTGTAGCTAATTTCTGTTTATGGAATC | 10–155 | 663 |
| URE3-BP 5' R | CTTGTATATTGATCTAATGGGATAGTGTTAAG | ||
| URE3-BP Middle F | GATGAGAATTTTTGATACTGATTTTAATGGAC | 276–454 | |
| URE3-BP Middle R | GATTAATATAGAATCCAAGTTGTTGAAGAG | ||
| URE3-BP 3' F | CTGTGATCTTAATTGTTGGATTG | 504–658 | |
| URE3-BP 3' R | CCAAGAGGGAAGTAACAACGT | ||
| Actin F | GCACTTGTTGTAGATAATGGATCAGGAATG | variable (detects all family members/alleles) | variable |
| Actin R | ACCCATACCAGCCATAACTGAAACG | ||
| Jacob F | CAAAGGAGTTCAAATGGGATGTGTTAG | variable (detects all family members/alleles) | variable |
| Jacob R | TTATTTGGTGTAGGAGTTGGTAATGGG | ||
Oligo pairs were designed to amplify short sections of Igl or URE3-BP. For Igl, four pairs of oligos were used: one amplifying the 5' end (Igl 5' oligo pair) and one the 3' end (Igl 3' oligo pair) of Igl1 and Igl2 simultaneously; and a pair each to amplify a short section unique to Igl1 or Igl2 (Igl1 oligo pair and Igl2 oligo pair, which have the same reverse primer in common) near the 5' end of the mRNA. Three oligo pairs were used to amplify short sections of URE3-BP: one pair the 5' end, one pair the middle, and one pair the 3' end. The actin and Jacob primers were designed to amplify all family members or alleles [35].