Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses
MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including <i>Cryptomeria fortunei,</i> has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR...
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MDPI AG
2021-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/14/7246 |
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| author | Yingting Zhang Jinyu Xue Lijuan Zhu Hailiang Hu Junjie Yang Jiebing Cui Jin Xu |
| author_facet | Yingting Zhang Jinyu Xue Lijuan Zhu Hailiang Hu Junjie Yang Jiebing Cui Jin Xu |
| author_sort | Yingting Zhang |
| collection | DOAJ |
| description | MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including <i>Cryptomeria fortunei,</i> has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (<i>U6</i>, <i>5S</i> and <i>18S</i>) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in <i>C. fortunei</i> under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and <i>U6</i> were the most stable RGs for studying <i>C. fortunei</i> miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of <i>C. fortunei</i> to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species. |
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| language | English |
| last_indexed | 2024-03-10T09:38:46Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f6b1b53692e54d8f9f9c672e500385e12023-11-22T03:55:26ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-07-012214724610.3390/ijms22147246Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple StressesYingting Zhang0Jinyu Xue1Lijuan Zhu2Hailiang Hu3Junjie Yang4Jiebing Cui5Jin Xu6Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaKey Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaKey Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaKey Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaKey Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaKey Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaKey Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, ChinaMicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including <i>Cryptomeria fortunei,</i> has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (<i>U6</i>, <i>5S</i> and <i>18S</i>) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in <i>C. fortunei</i> under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and <i>U6</i> were the most stable RGs for studying <i>C. fortunei</i> miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of <i>C. fortunei</i> to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species.https://www.mdpi.com/1422-0067/22/14/7246miRNAsreference geneqRT-PCRabiotic stresshormone treatmenttissue |
| spellingShingle | Yingting Zhang Jinyu Xue Lijuan Zhu Hailiang Hu Junjie Yang Jiebing Cui Jin Xu Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses International Journal of Molecular Sciences miRNAs reference gene qRT-PCR abiotic stress hormone treatment tissue |
| title | Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses |
| title_full | Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses |
| title_fullStr | Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses |
| title_full_unstemmed | Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses |
| title_short | Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (<i>Cryptomeria fortunei</i>) under Multiple Stresses |
| title_sort | selection and optimization of reference genes for microrna expression normalization by qrt pcr in chinese cedar i cryptomeria fortunei i under multiple stresses |
| topic | miRNAs reference gene qRT-PCR abiotic stress hormone treatment tissue |
| url | https://www.mdpi.com/1422-0067/22/14/7246 |
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