Validation of reference genes for accurate normalization of gene expression with quantitative real-time PCR in Haloxylon ammodendron under different abiotic stresses

Abstract

plays an important role in maintaining the structure and function of the entire ecosystem where it grows. No suitable reference genes have been reported in plants to date. In this study, a total of 8 reference genes (, ,, ,, ,- and ) were selected from the available trancriptome database, and the expression stability of these 8 candidate genes was validated under different abiotic stress with three different statistical algorithms (geNorm, NormFinder and BestKeeper). The results produced from different models were in agreement with each other essentially: and were the most stable genes under drought stress, the most stable gene under heat stress and mechanical damage, and stable under salt stress while and expressed constantly under mechanical damage, and expressed steadily under cold conditions. Expression profiles of several stress response genes, including-, -, and, were further confirmed with various candidate reference genes. None of the candidate genes showed a constant expression among all tested samples. Hence, it's essential to use more than one reference gene in order to guarantee the accuracy of quantitative real-time PCR. The results of this study will contribute to the accuracy and reliability in transcripts quantification, which is of significance to transcription-based studies and applications in this important shrub .

Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

“…Selection of reference genes and verification of primer specificity. We selected 10 genes as candidate genes by mining the N. tangutorum transcriptome data according to previous reports on the selection of reference genes in model plants and non-model plants [13][14][15][16][17][18][19][20][21][22] . The chosen N. tangutorum genes were then used to query against the Arabidopsis protein database using BLASTX to obtain the homologous genes.…”

“…However, with our increasing understanding of the agricultural ecological value of non-model plants, it has become important to study their molecular genetic mechanisms and to determine their appropriate reference genes under different experimental conditions. To date, some non-model plants have been selected for research on internal reference genes, including Reaumuria 16 , Caragana intermedia 17 , Setaria viridis 18 , Miscanthus lutarioriparia 19 , Haloxylon ammodendron 20 , Hylocereus undulatus Britt 21 , and Corchorus capsularis L 22 .…”

Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid

“…This difference in reference gene evaluation could also be seen in Undaria pinnatifida [35]. However, studies have also been reported in which NormFinder and BestKeeper provided similar outcomes [36]. Multiple control reference genes are essential if possible [10].…”

Selection and validation of reference genes for quantitative real-time PCR analysis of Nitraria tangutorum

“…The quantitative primers of the candidate reference genes were designed by the Primer3Plus online website (www.primer3plus.com/cgi-bin/dev/primer3plus.cgi). The parameters were as follows: the length of the primers and the amplification product size were [18][19][20][21][22][23][24][25][26][27] bp and 100-200 bp, respectively, the range of melting temperature (Tm) was 65°C-75°C, and the GC was 40%-60%. Primers were synthesized by Sangon Biotech.…”

“…it has become important to study their molecular genetic mechanism, and to determine their appropriate reference genes under different experimental conditions. To date, some non-model plants have been selected for internal reference genes, including Reaumuria [16], Caragana intermedia [17], Setaria viridis [18], Miscanthus lutarioriparia [19], Haloxylon ammodendron [20], pitaya [21], and jute [22].…”