Table 1. Descriptions of mRNAs and miRNAs and RT-qPCR amplification characteristics.
| Symbol | Name | Annotation | Accession No | Primer design | Amplicon length(bp) | Amplicon Tm(°C) | Regression coefficient (R2) | PCR efficiency (%) |
|---|---|---|---|---|---|---|---|---|
| Candidate reference mRNA genes for microRNA and miRNA precursor normalization | ||||||||
| Act | Actin | Cytoskeletal structural protein | (phytozome)Glyma.02G091900.1 | F: GACCTTCAACACCCCTGCT | 143bp | 85.0°C | 1.000 | 101.8% |
| R: GTGGGAGTGCATAACCCTC | ||||||||
| Cyp | Cyclophilin | Cyclophilin type peptidyl- prolyl cis-trans isomerase activity | (phytozome)Glyma.12G024700.1 | F: TCCGAGCACCGCCGAGAACT | 135bp | 91.0°C | 0.999 | 99.8% |
| R: AAGTCGCCGCCCTGGCACAT | ||||||||
| EF1a | Elongation Factor 1-alpha | translation elongation factor | (phytozome)Glyma.19G052400.1 | F: GCTCTTCTTGCTTTCACCCTT | 111bp | 82.5°C | 0.999 | 106.8% |
| R: TTCCTTCACAATTTCATCATACC | ||||||||
| EF1b | Elongation Factor1- beta | translation elongation factor | (phytozome)Glyma.14G039100.1 | F: TGGTGATGAGACAGAGGAGGA | 106bp | 83.0°C | 1.000 | 98.1% |
| R: AACATCGAGAAGGACAGAAGA | ||||||||
| Fbox | F-box | F-box protein | (phytozome)Glyma.12G051100.1 | F: CATCCGTGCTAATGATATTGT | 127bp | 83.5°C | 0.996 | 107.8% |
| R: TGATGGTGTTGGTAGAGGC | ||||||||
| TuB | Beta Tubulin | Structural constituent of cytoskeleton | (phytozome)Glyma.19G127700.1 | F: CCAGTTGGTGGAGAATGCT | 129bp | 86.0°C | 0.996 | 92.9% |
| R: ATGGTTGCGGAGATCAAGTGA | ||||||||
| TuA | Alpha Tubulin | Structural constituent of cytoskeleton | (phytozome)Glyma.05G157300.1 | F: TTGCCACCATCAAGACTAAGA | 104bp | 86.0°C | 0.998 | 95.9% |
| R: ACCACCAGGAACAACAGAAG | ||||||||
| 60s | 60s ribosomal | 60s ribosomal protein | (phytozome)Glyma.13G318800.1 | F: GTCCTGAAGGCCATTCCTAAG | 132bp | 82.5°C | 0.998 | 100% |
| R: ACTGATGCCCTGCTCCAACT | ||||||||
| Candidate reference miRNAs for mature microRNA normalization | ||||||||
| 156a | Mature gma-miR156a-5p | Mature miRNA | (miRBase) MIMAT0001686 | F: TGACAGAAGAGAGTGAGCACA | - b | 78.5°C | 0.999 | 108.8% |
| R: Universal RT Primera | ||||||||
| 166a | Mature gma-miR166a-3p | Mature miRNA | (miRBase)MIMAT0001677 | F: GGACCAGGCTTCATTCCCCA | - | 80.5°C | 0.999 | 97.1% |
| R: Universal RT Primer | ||||||||
| 167a | Mature gma-miR167a-5p | Mature miRNA | (miRBase) MIMAT0001679 | F: TGAAGCTGCCAGCATGATCTA | - | 79.0°C | 0.999 | 94.5% |
| R: Universal RT Primer | ||||||||
| 171a | Mature gma-miR171a-3p | Mature miRNA | (miRBase)MIMAT0007358 | F: TGAGCCGTGCCAATATCACGA | - | 79.5°C | 0.998 | 107.5% |
| R: Universal RT Primer | ||||||||
| 172a | Mature gma-miR172a-3p | Mature miRNA | (miRBase) MIMAT0001682 | F: GAGAATCTTGATGATGCTGCAT | - | 78.0°C | 0.999 | 100.3% |
| R: Universal RT Primer | ||||||||
| 393a | Mature gma-miR393a-5p | Mature miRNA | (miRBase)MIMAT0007362 | F: TCCAAAGGGATCGCATTGATCA | - | 78.5°C | 0.993 | 100.1% |
| R: Universal RT Primer | ||||||||
| 397a | Mature gma-miR397a-5p | Mature miRNA | (miRBase) MIMAT0021627 | F: TCATTGAGTGCAGCGTTGATGA | - | 79.0°C | 0.998 | 104.7% |
| R: Universal RT Primer | ||||||||
| 1520d | Mature gma-miR1520d-3p | Mature miRNA | (miRBase) MIMAT0007379 | F: ATCAGAACATGACACGTGACAA | - | 78.5°C | 0.991 | 108.7% |
| R: Universal RT Primer | ||||||||
| Reference mRNA and miRNA validation | ||||||||
| 396a | Mature gma-miR396a-5p | Mature miRNA | (miRBase)MIMAT0001687 | F: TTCCACAGCTTTCTTGAACTGA | - | 78.5°C | 0.998 | 98% |
| R: Universal RT Primer | ||||||||
| Pre-396a | Precursors gma-miR396a | Mature precursors | (miRBase)MI0001785 | F: GCTTTCTTGAACTGCATCCAA | 94bp | 81.0°C | 0.999 | 101.6% |
| R: TCCCACAGCTTTATTGAACCG | ||||||||
| GRF9 | Growth Regulating Factor 9 | Transcription factors | (phytozome)Glyma.01G144900.1 | F: GATGGAAAGAAATGGAGGTG | 137bp | 79.0°C | 0.999 | 107.3% |
| R: GGCTTTGTTGCCAGATGAG | ||||||||
amiRNA universal reverse primers were provided in the NCode VILO miRNA cDNA Synthesis Kit (Invitrogen).
bAmplicon length of the miRNA precursors cannot be determined.