Primer Sequences and Amplification Efficiencies of Evaluated Normalizers for Singleplex Assays and miRNAs Used in Validation Cohort
| Assay Name . | miRBase Accession No. . | miRBase Name . | Target Sequence (5′→3′) . | Slope . | PEA . | EA . | DR . | R2 . | Selected as Normalizer . |
|---|---|---|---|---|---|---|---|---|---|
| miR-16 | MIMAT0000069 | hsa-mir-16-5p | UAGCAGCACGUAAAUAUUGGCG | −4.35 | 76% | 1.6978 | 5 | 0.991 | Jia et al., 2013 (16) |
| miR-28-p | MIMAT0000085 | hsa-mir-28-5p | AAGGAGCUCACAGUCUAUUGAG | −3.77 | 88% | 1.8430 | 4 | 0.996 | GeNorm (our experiment) |
| miR-30a | MIMAT0000087 | hsa-mir-30a-5p | TGTAAACATCCTCGACTGGAAG | −3.89 | 86% | 1.8088 | 4 | 0.998 | GeNorm (our experiment) |
| miR-30b | MIMAT0000420 | hsa-mir-30b-5p | UGUAAACAUCCUACACUCAGCU | −3.76 | 88% | 1.8442 | 5 | 0.996 | GeNorm (our experiment) |
| miR-30d | MIMAT0000245 | hsa-mir-30d-5p | TGTAAACATCCCCGACTGGAAG | −3.89 | 85% | 1.8080 | 4 | 0.997 | GeNorm (our experiment) |
| miR-30e | MIMAT0000692 | hsa-mir-30e-5p | TGTAAACATCCTTGACTGGAAG | −3.87 | 86% | 1.8136 | 4 | 0.998 | GeNorm (our experiment), |
| Rekker et al., 2015 (40) | |||||||||
| miR-132 | MIMAT0000426 | hsa-mir-132-3p | UAACAGUCUACAGCCAUGGUCG | −3.22 | 103% | 2.0430 | 5 | 0.992 | Suryawanshi et al., 2013 (17) |
| RNU6B | NR_002752 | sn rna U6 (RNU6B) | CGCAAGGATGACACGCAAATTCGTGAAGCGTTCCATATTTTT | −2.89 | 115% | 2.2183 | 2 | 0.999 | Others |
| miRNAs used in validation cohort | |||||||||
| miR-9* | MIMAT0000442 | hsa-mir-9-3p | ATAAAGCTAGATAACCGAAAGT | −3.213 | 103% | 2.0476 | 3 | 0.998 | |
| miR-29c | MIMAT0000681 | hsa-mir-29c-3p | UAGCACCAUUUGAAAUCGGUUA | −4.19 | 79% | 1.7325 | 5 | 0.995 | |
| miR-135b | MIMAT0000758 | hsa-mir-135b-5p | UAUGGCUUUUCAUUCCUAUGUGA | −3.148 | 106% | 2.0781 | 2 | 0.964 | |
| miR-139-3p | MIMAT0004552 | hsa-mir-139-3p | GGAGACGCGGCCCUGUUGGAGU | −3.887 | 86% | 1.8083 | 3 | 0.996 | |
| miR-141* | MIMAT0004598 | hsa-mir-141-5p | CATCTTCCAGTACAGTGTTGGA | −2.655 | 125% | 2.3804 | 2 | 0.883 | |
| miR-145* | MIMAT0004601 | hsa-mir-145-3p | GGATTCCTGGAAATACTGTTCT | −3.372 | 99% | 1.9795 | 3 | 0.996 | |
| miR-155 | MIMAT0000646 | hsa-mir-155-5p | UUAAUGCUAAUCGUGAUAGGGGU | −4.639 | 72% | 1.6427 | 5 | 0.905 | |
| miR-574-3p | MIMAT0003239 | hsa-mir-574-3p | CACGCUCAUGCACACACCCACA | −3.412 | 97% | 1.9637 | 5 | 0.986 | |
| miR-923 | MI0005715 | hsa-mir-923 | GTCAGCGGAGGAAAAGAAACT | −4.450 | 75% | 1.6777 | 4 | 0.995 | |
| Assay Name . | miRBase Accession No. . | miRBase Name . | Target Sequence (5′→3′) . | Slope . | PEA . | EA . | DR . | R2 . | Selected as Normalizer . |
|---|---|---|---|---|---|---|---|---|---|
| miR-16 | MIMAT0000069 | hsa-mir-16-5p | UAGCAGCACGUAAAUAUUGGCG | −4.35 | 76% | 1.6978 | 5 | 0.991 | Jia et al., 2013 (16) |
| miR-28-p | MIMAT0000085 | hsa-mir-28-5p | AAGGAGCUCACAGUCUAUUGAG | −3.77 | 88% | 1.8430 | 4 | 0.996 | GeNorm (our experiment) |
| miR-30a | MIMAT0000087 | hsa-mir-30a-5p | TGTAAACATCCTCGACTGGAAG | −3.89 | 86% | 1.8088 | 4 | 0.998 | GeNorm (our experiment) |
| miR-30b | MIMAT0000420 | hsa-mir-30b-5p | UGUAAACAUCCUACACUCAGCU | −3.76 | 88% | 1.8442 | 5 | 0.996 | GeNorm (our experiment) |
| miR-30d | MIMAT0000245 | hsa-mir-30d-5p | TGTAAACATCCCCGACTGGAAG | −3.89 | 85% | 1.8080 | 4 | 0.997 | GeNorm (our experiment) |
| miR-30e | MIMAT0000692 | hsa-mir-30e-5p | TGTAAACATCCTTGACTGGAAG | −3.87 | 86% | 1.8136 | 4 | 0.998 | GeNorm (our experiment), |
| Rekker et al., 2015 (40) | |||||||||
| miR-132 | MIMAT0000426 | hsa-mir-132-3p | UAACAGUCUACAGCCAUGGUCG | −3.22 | 103% | 2.0430 | 5 | 0.992 | Suryawanshi et al., 2013 (17) |
| RNU6B | NR_002752 | sn rna U6 (RNU6B) | CGCAAGGATGACACGCAAATTCGTGAAGCGTTCCATATTTTT | −2.89 | 115% | 2.2183 | 2 | 0.999 | Others |
| miRNAs used in validation cohort | |||||||||
| miR-9* | MIMAT0000442 | hsa-mir-9-3p | ATAAAGCTAGATAACCGAAAGT | −3.213 | 103% | 2.0476 | 3 | 0.998 | |
| miR-29c | MIMAT0000681 | hsa-mir-29c-3p | UAGCACCAUUUGAAAUCGGUUA | −4.19 | 79% | 1.7325 | 5 | 0.995 | |
| miR-135b | MIMAT0000758 | hsa-mir-135b-5p | UAUGGCUUUUCAUUCCUAUGUGA | −3.148 | 106% | 2.0781 | 2 | 0.964 | |
| miR-139-3p | MIMAT0004552 | hsa-mir-139-3p | GGAGACGCGGCCCUGUUGGAGU | −3.887 | 86% | 1.8083 | 3 | 0.996 | |
| miR-141* | MIMAT0004598 | hsa-mir-141-5p | CATCTTCCAGTACAGTGTTGGA | −2.655 | 125% | 2.3804 | 2 | 0.883 | |
| miR-145* | MIMAT0004601 | hsa-mir-145-3p | GGATTCCTGGAAATACTGTTCT | −3.372 | 99% | 1.9795 | 3 | 0.996 | |
| miR-155 | MIMAT0000646 | hsa-mir-155-5p | UUAAUGCUAAUCGUGAUAGGGGU | −4.639 | 72% | 1.6427 | 5 | 0.905 | |
| miR-574-3p | MIMAT0003239 | hsa-mir-574-3p | CACGCUCAUGCACACACCCACA | −3.412 | 97% | 1.9637 | 5 | 0.986 | |
| miR-923 | MI0005715 | hsa-mir-923 | GTCAGCGGAGGAAAAGAAACT | −4.450 | 75% | 1.6777 | 4 | 0.995 | |
The percentage of amplification efficiency (PEA) was determined as (log 2 EA) × 100% [EA = 2^PEA], where amplification efficiency (EA) is 10−1/slope.
Abbreviations: DR, dynamic range; R2, coefficient of determination in regression model.
Primer Sequences and Amplification Efficiencies of Evaluated Normalizers for Singleplex Assays and miRNAs Used in Validation Cohort
| Assay Name . | miRBase Accession No. . | miRBase Name . | Target Sequence (5′→3′) . | Slope . | PEA . | EA . | DR . | R2 . | Selected as Normalizer . |
|---|---|---|---|---|---|---|---|---|---|
| miR-16 | MIMAT0000069 | hsa-mir-16-5p | UAGCAGCACGUAAAUAUUGGCG | −4.35 | 76% | 1.6978 | 5 | 0.991 | Jia et al., 2013 (16) |
| miR-28-p | MIMAT0000085 | hsa-mir-28-5p | AAGGAGCUCACAGUCUAUUGAG | −3.77 | 88% | 1.8430 | 4 | 0.996 | GeNorm (our experiment) |
| miR-30a | MIMAT0000087 | hsa-mir-30a-5p | TGTAAACATCCTCGACTGGAAG | −3.89 | 86% | 1.8088 | 4 | 0.998 | GeNorm (our experiment) |
| miR-30b | MIMAT0000420 | hsa-mir-30b-5p | UGUAAACAUCCUACACUCAGCU | −3.76 | 88% | 1.8442 | 5 | 0.996 | GeNorm (our experiment) |
| miR-30d | MIMAT0000245 | hsa-mir-30d-5p | TGTAAACATCCCCGACTGGAAG | −3.89 | 85% | 1.8080 | 4 | 0.997 | GeNorm (our experiment) |
| miR-30e | MIMAT0000692 | hsa-mir-30e-5p | TGTAAACATCCTTGACTGGAAG | −3.87 | 86% | 1.8136 | 4 | 0.998 | GeNorm (our experiment), |
| Rekker et al., 2015 (40) | |||||||||
| miR-132 | MIMAT0000426 | hsa-mir-132-3p | UAACAGUCUACAGCCAUGGUCG | −3.22 | 103% | 2.0430 | 5 | 0.992 | Suryawanshi et al., 2013 (17) |
| RNU6B | NR_002752 | sn rna U6 (RNU6B) | CGCAAGGATGACACGCAAATTCGTGAAGCGTTCCATATTTTT | −2.89 | 115% | 2.2183 | 2 | 0.999 | Others |
| miRNAs used in validation cohort | |||||||||
| miR-9* | MIMAT0000442 | hsa-mir-9-3p | ATAAAGCTAGATAACCGAAAGT | −3.213 | 103% | 2.0476 | 3 | 0.998 | |
| miR-29c | MIMAT0000681 | hsa-mir-29c-3p | UAGCACCAUUUGAAAUCGGUUA | −4.19 | 79% | 1.7325 | 5 | 0.995 | |
| miR-135b | MIMAT0000758 | hsa-mir-135b-5p | UAUGGCUUUUCAUUCCUAUGUGA | −3.148 | 106% | 2.0781 | 2 | 0.964 | |
| miR-139-3p | MIMAT0004552 | hsa-mir-139-3p | GGAGACGCGGCCCUGUUGGAGU | −3.887 | 86% | 1.8083 | 3 | 0.996 | |
| miR-141* | MIMAT0004598 | hsa-mir-141-5p | CATCTTCCAGTACAGTGTTGGA | −2.655 | 125% | 2.3804 | 2 | 0.883 | |
| miR-145* | MIMAT0004601 | hsa-mir-145-3p | GGATTCCTGGAAATACTGTTCT | −3.372 | 99% | 1.9795 | 3 | 0.996 | |
| miR-155 | MIMAT0000646 | hsa-mir-155-5p | UUAAUGCUAAUCGUGAUAGGGGU | −4.639 | 72% | 1.6427 | 5 | 0.905 | |
| miR-574-3p | MIMAT0003239 | hsa-mir-574-3p | CACGCUCAUGCACACACCCACA | −3.412 | 97% | 1.9637 | 5 | 0.986 | |
| miR-923 | MI0005715 | hsa-mir-923 | GTCAGCGGAGGAAAAGAAACT | −4.450 | 75% | 1.6777 | 4 | 0.995 | |
| Assay Name . | miRBase Accession No. . | miRBase Name . | Target Sequence (5′→3′) . | Slope . | PEA . | EA . | DR . | R2 . | Selected as Normalizer . |
|---|---|---|---|---|---|---|---|---|---|
| miR-16 | MIMAT0000069 | hsa-mir-16-5p | UAGCAGCACGUAAAUAUUGGCG | −4.35 | 76% | 1.6978 | 5 | 0.991 | Jia et al., 2013 (16) |
| miR-28-p | MIMAT0000085 | hsa-mir-28-5p | AAGGAGCUCACAGUCUAUUGAG | −3.77 | 88% | 1.8430 | 4 | 0.996 | GeNorm (our experiment) |
| miR-30a | MIMAT0000087 | hsa-mir-30a-5p | TGTAAACATCCTCGACTGGAAG | −3.89 | 86% | 1.8088 | 4 | 0.998 | GeNorm (our experiment) |
| miR-30b | MIMAT0000420 | hsa-mir-30b-5p | UGUAAACAUCCUACACUCAGCU | −3.76 | 88% | 1.8442 | 5 | 0.996 | GeNorm (our experiment) |
| miR-30d | MIMAT0000245 | hsa-mir-30d-5p | TGTAAACATCCCCGACTGGAAG | −3.89 | 85% | 1.8080 | 4 | 0.997 | GeNorm (our experiment) |
| miR-30e | MIMAT0000692 | hsa-mir-30e-5p | TGTAAACATCCTTGACTGGAAG | −3.87 | 86% | 1.8136 | 4 | 0.998 | GeNorm (our experiment), |
| Rekker et al., 2015 (40) | |||||||||
| miR-132 | MIMAT0000426 | hsa-mir-132-3p | UAACAGUCUACAGCCAUGGUCG | −3.22 | 103% | 2.0430 | 5 | 0.992 | Suryawanshi et al., 2013 (17) |
| RNU6B | NR_002752 | sn rna U6 (RNU6B) | CGCAAGGATGACACGCAAATTCGTGAAGCGTTCCATATTTTT | −2.89 | 115% | 2.2183 | 2 | 0.999 | Others |
| miRNAs used in validation cohort | |||||||||
| miR-9* | MIMAT0000442 | hsa-mir-9-3p | ATAAAGCTAGATAACCGAAAGT | −3.213 | 103% | 2.0476 | 3 | 0.998 | |
| miR-29c | MIMAT0000681 | hsa-mir-29c-3p | UAGCACCAUUUGAAAUCGGUUA | −4.19 | 79% | 1.7325 | 5 | 0.995 | |
| miR-135b | MIMAT0000758 | hsa-mir-135b-5p | UAUGGCUUUUCAUUCCUAUGUGA | −3.148 | 106% | 2.0781 | 2 | 0.964 | |
| miR-139-3p | MIMAT0004552 | hsa-mir-139-3p | GGAGACGCGGCCCUGUUGGAGU | −3.887 | 86% | 1.8083 | 3 | 0.996 | |
| miR-141* | MIMAT0004598 | hsa-mir-141-5p | CATCTTCCAGTACAGTGTTGGA | −2.655 | 125% | 2.3804 | 2 | 0.883 | |
| miR-145* | MIMAT0004601 | hsa-mir-145-3p | GGATTCCTGGAAATACTGTTCT | −3.372 | 99% | 1.9795 | 3 | 0.996 | |
| miR-155 | MIMAT0000646 | hsa-mir-155-5p | UUAAUGCUAAUCGUGAUAGGGGU | −4.639 | 72% | 1.6427 | 5 | 0.905 | |
| miR-574-3p | MIMAT0003239 | hsa-mir-574-3p | CACGCUCAUGCACACACCCACA | −3.412 | 97% | 1.9637 | 5 | 0.986 | |
| miR-923 | MI0005715 | hsa-mir-923 | GTCAGCGGAGGAAAAGAAACT | −4.450 | 75% | 1.6777 | 4 | 0.995 | |
The percentage of amplification efficiency (PEA) was determined as (log 2 EA) × 100% [EA = 2^PEA], where amplification efficiency (EA) is 10−1/slope.
Abbreviations: DR, dynamic range; R2, coefficient of determination in regression model.
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