Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Candidate miRNA References Selection
2.2.1. miRNA-Seq for Normal Tissues and Bacterial Infected Livers of Japanese Flounder
2.2.2. Published miRNA References for Teleost Species
2.2.3. Commonly Used miRNA References for qRT-PCR: U6, 5S rRNA, and 18S rRNA
2.3. RNA Extraction and miRNA cDNA Synthesis
2.4. qRT-PCR Analysis
2.5. Statistical Analysis
2.6. Reference miRNA Validation
3. Results
3.1. Candidate Reference Selection for Japanese Flounder miRNA Quantification
3.2. Amplification Efficiency of the Candidate References by qRT-PCR
3.3. Expression Stability of the Candidate References among Normal Tissues of Japanese Flounder by qRT-PCR
3.4. Expression Stability of Candidate References in Japanese Flounder Livers Injected with E. tarda or Ringer’s Solution by qRT-PCR
3.5. Reference miRNA Validation in Japanese Flounder Livers Infected with E. tarda by qRT-PCR
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Genes | Primer Sequence (5′–3′) | Amplification Efficiency |
---|---|---|
miR-34a-5p-F | TGGCAGTGTCTTAGCTGGTTGT | 173.84% |
miR-205-5p-F | TCCTTCATTCCACCGGAGTCTG | 90.74% |
miR-101a-3p-F | TACAGTACTGTGATAACTGAAG | 96.22% |
miR-22-3p-F | AAGCTGCCAGCTGAAGAACTGT | 99.98% |
miR-23a-3p-F | ATCACATTGCCAGGGATTTCCA | 110.00% |
miR-210-5p-F | AGCCACTGACTAACGCACATTG | 131.01% |
miR-30c-5p-F | TGTAAACATCCTTGACTGGAAGCT | 220% |
5S rRNA-F | CCATACCACCCTGAACAC | 83.38% |
5S rRNA-R | CGGTCTCCCATCCAAGTA | |
18S rRNA-F | CCTGAGAAACGGCTACCACAT | 85.45% |
18S rRNA-R | CCAATTACAGGGCCTCGAAAG | |
U6-F | TTGGAACGATACAGAGAAGATTAGC | 86.42% |
Method | Ranking Order (Better–Good–Average) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Delta CT | miR-22-3p | miR-23a-3p | miR-101a-3p | miR-30c-5p | miR-34a-5p | miR-210-5p | 18S rRNA | miR-205-5p | U6 | 5S rRNA |
BestKeeper | miR-34a-5p | miR-101a-3p | miR-22-3p | miR-30c-5p | miR-210-5p | miR-23a-3p | 18S rRNA | U6 | miR-205-5p | 5S rRNA |
NormFinder | miR-22-3p | miR-210-5p | miR-23a-3p | miR-30c-5p | miR-101a-3p | 18S rRNA | miR-34a-5p | miR-205-5p | U6 | 5S rRNA |
geNorm | miR-22-3p|miR-23a-3p | miR-101a-3p | miR-34a-5p | miR-30c-5p | miR-210-5p | 18S rRNA | miR-205-5p | U6 | 5S rRNA | |
Recommended comprehensive ranking | miR-22-3p | miR-23a-3p | miR-101a-3p | miR-34a-5p | miR-30c-5p | miR-210-5p | 18S rRNA | miR-205-5p | U6 | 5S rRNA |
geNorm | miR-22-3p|miR-23a-3p | miR-101a-3p | miR-34a-5p | miR-30c-5p | miR-210-5p | 18S rRNA | miR-205-5p | U6 | 5S rRNA |
---|---|---|---|---|---|---|---|---|---|
geNorm Stability value (M) | 0.652 | 0.770 | 0.899 | 0.997 | 1.123 | 1.263 | 1.493 | 1.736 | 2.514 |
Method | Ranking Order (Better--Good--Average) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Delta CT | miR-210-5p | miR-22-3p | miR-23a-3p | miR-205-5p | miR-30c-5p | 18S rRNA | miR-34a-5p | U6 | miR-101a-3p | 5S rRNA |
BestKeeper | 5S rRNA | miR-101a-3p | miR-34a-5p | miR-30c-5p | miR-210-5p | 18S rRNA | miR-205-5p | miR-22-3p | miR-23a-3p | U6 |
NormFinder | miR-210-5p | miR-22-3p | miR-23a-3p | miR-205-5p | miR-30c-5p | 18S rRNA | miR-34a-5p | U6 | miR-101a-3p | 5S rRNA |
geNorm | miR-22-3p|miR-23a-3p | miR-210-5p | 18S rRNA | miR-205-5p | miR-30c-5p | U6 | miR-34a-5p | miR-101a-3p | 5S rRNA | |
Recommended comprehensive ranking | miR-210-5p | miR-22-3p | miR-23a-3p | miR-205-5p | miR-30c-5p | 18S rRNA | miR-34a-5p | 5S rRNA | miR-101a-3p | U6 |
geNorm | miR-22-3p|miR-23a-3p | miR-210-5p | 18S rRNA | miR-205-5p | miR-30c-5p | U6 | miR-34a-5p | miR-101a-3p | 5S rRNA |
---|---|---|---|---|---|---|---|---|---|
geNorm Stability value (M) | 0.292 | 0.356 | 0.622 | 0.811 | 0.942 | 1.102 | 1.284 | 1.396 | 1.510 |
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Liu, S.; Song, H.; Liu, Z.; Lu, W.; Zhang, Q.; Cheng, J. Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers. Genes 2022, 13, 175. https://doi.org/10.3390/genes13020175
Liu S, Song H, Liu Z, Lu W, Zhang Q, Cheng J. Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers. Genes. 2022; 13(2):175. https://doi.org/10.3390/genes13020175
Chicago/Turabian StyleLiu, Saisai, Haofei Song, Zeyu Liu, Wei Lu, Quanqi Zhang, and Jie Cheng. 2022. "Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers" Genes 13, no. 2: 175. https://doi.org/10.3390/genes13020175
APA StyleLiu, S., Song, H., Liu, Z., Lu, W., Zhang, Q., & Cheng, J. (2022). Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers. Genes, 13(2), 175. https://doi.org/10.3390/genes13020175