TABLE 1.
Oligonucleotides used in the Invader RNA assay
| Probe set | Oligonucleotide | Sequencea (5′ to 3′) |
|---|---|---|
| aLowercase bases indicate 5′ flap sequences. Cleavage base indicated bold. A 3′NH2 can be included to enhance assay performance. Underlined bases indicate 2` O-methylated nucleotides. | ||
| Redmond Red (Red) and Eclipse Dark Quencher (DQ) were obtained from Epoch Biosciences (Bothell, WA). | ||
| U-D | Probe | 5′-ccgtcgctgcgtCTGGTAGGGT-NH2-3′ |
| Invader oligo | 5′-TCCTGCAGCTCAACCGCGACA-3′ | |
| Stacking oligo | 5′-ACCUCGCAGACAGCG-3′ | |
| Arrestor oligo | 5′-ACCCUACCAGACGCAG-3′ | |
| FRET oligo | FAM-5′-CAC(DQ)TGCTTCGTGG-3′ | |
| Secondary reaction template | 5′-CCAGGAAGCAAGTGACGCAGCGACGGU-3′ | |
| Synthetic target oligo (coding) | 5′-GGTAATACGACTCACTATAGGGACGCATCGCTGTCTGCGAGGTACCCTACCAGGTCGCGGTTGAGCTGCAGGACAAAC-3′ | |
| Synthetic target oligo (noncoding) | 5′-GTTTGTCCTGCAGCTCAACCGCGACCTGGTAGGGTACCTCGCAGACAGCGATGCGTCCCTATAGTGAGTCGTATTACC-3′ | |
| III-D | Probe | 5′-ccgtcacgcctcCTTGCTGTTTG-NH2-3′ |
| Invader oligo | 5′-TCCTGCAGCTCAACCGCGACA-3′ | |
| Stacking oligo | 5′-GGCAGGACAGUGAGCC-3′ | |
| Arrestor oligo | 5′-CAAACAGCAAGGAGGCG-3′ | |
| FRET oligo | Red-5′-CTC(DQ)TTCTCAGTGCG-3′ | |
| Secondary reaction template | 5′-CGCAGTGAGAATGAGGAGGCGTGACGGU-3′ | |
| Synthetic target oligo (coding) | 5′-GGTAATACGACTCACTATAGGGATGCCTGGCTCACTGTCCTGCCCAAACAGCAAGGTCGCGGTTGAGCTGCAGGACAAAC-3′ | |
| Synthetic target oligo (noncoding) | 5′-GTTTGTCCTGCAGCTCAACCGCGACCTTGCTGTTTGGGCAGGACAGTGAGCCAGGCATCCCTATAGTGAGTCGTATTACC-3′ | |
| U-IIIc | Probe | 5′-ccgtcgctgcgtCTGGTAGGGT-NH2-3′ |
| Invader oligo | 5′-CGTGGTGTTAACACCGGCGGCA-3′ | |
| Stacking oligo | 5′-ACCUCGCAGACAGCG-3′ | |
| Arrestor oligo | 5′-ACCCUACCAGACGCAG-3′ | |
| FRET oligo | FAM-5′-CAC(DQ)TGCTTCGTGG-3′ | |
| Secondary reaction template | 5′-CCAGGAAGCAAGTGACGCAGCGACGGU-3′ | |
| Synthetic target oligo (coding) | 5′-GGTAATACGACTCACTATAGGGACGCATCGCTGTCTGCGAGGTACCCTACCAGGCCGCCGGTGTTAACACCACGGACAA-3′ | |
| Synthetic target oligo (noncoding) | 5′-TTGTCCGTGGTGTTAACACCGGCGGCCTGGTAGGGTACCTCGCAGACAGCGATGCGTCCCTATAGTGAGTCGTATTACC-3′ | |
| IIIb-IIIc | Probe | 5′-ccgtcacgcctcCTTGCTGTTTG-NH2-3′ |
| Invader oligo | 5′-CGTGGTGTTAACACCGGCGGCA-3′ | |
| Stacking oligo | 5′-GGCAGGACAGUGAGCC-3′ | |
| Arrestor oligo | 5′-CAAACAGCAAGGAGGCG-3′ | |
| FRET oligo | Red-5′-CTC(DQ)TTCTCAGTGCG-3′ | |
| Secondary reaction template | 5′-CGCAGTGAGAATGAGGAGGCGTGACGGU-3′ | |
| Synthetic target oligo (coding) | 5′-AGGTAATACGACTCACTATAGGGATGCCTGGCTCACTGTCCTGCCCAAACAGCAAGGCCGCCGGTGTTAACACCACGGACAAT-3′ | |
| Synthetic target oligo (noncoding) | 5′-ATTGTCCGTGGTGTTAACACCGGCGGCCTTGCTGTTTGGGCAGGACAGTGAGCCAGGCATCCCTATAGTGAGTCGTATTACCT-3′ | |
| IIIc-D | Probe | 5′-ccgtcacgcctcCTGGCAGAAC-NH2-3′ |
| Invader oligo | 5′-TCCTGCAGCTCAACCGCGACA-3′ | |
| Stacking oligo | 5′-UGUCAACCAUGCAGAGU-3′ | |
| Arrestor oligo | 5′-GUUCUGCCAGGAGGCG-3′ | |
| FRET oligo | Red-5′-CTC(DQ)TTCTCAGTGCG-3′ | |
| Secondary reaction template | 5′-CGCAGTGAGAATGAGGAGGCGTGACGGU-3′ | |
| Synthetic target oligo (coding) | 5′-GGTAATACGACTCACTATAGGGACCTTTCACTCTGCATGGTTGACAGTTCTGCCAGGTCGCGGTTGAGCTGCAGGACAAAC-3′ | |
| Synthetic target oligo (noncoding) | 5′-GTTTGTCCTGCAGCTCAACCGCACCTGGCAGAACTGTCAACCATGCAGAGTGAAAGGTCCCTATAGTGAGTCGTATTACC-3′ | |
