Dataset Information

Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.

ABSTRACT:

Background

Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro.

Results

In this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1? were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants.

Conclusions

geNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1? had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.

SUBMITTER: Liu M

PROVIDER: S-EPMC3911976 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.

Liu Mingying M Jiang Jing J Han Xiaojiao X Qiao Guirong G Zhuo Renying R

PloS one 20140203 2

<h4>Background</h4>Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference ...[more]

PMID: 24498321

Similar Datasets

Project description:Ma bamboo (Dendrocalamus latiflorus Munro) is the most widely cultivated clumping bamboo in Southern China and is valuable for both consumption and wood production. The development of bamboo shoots involving the occurrence of lateral buds is unique, and it affects both shoot yield and the resulting timber. Plant-specific TCP transcription factors are involved in plant growth and development, particularly in lateral bud outgrowth and morphogenesis. However, the comprehensive information of the TCP genes in Ma bamboo remains poorly understood. In this study, 66 TCP transcription factors were identified in Ma bamboo at the genome-wide level. Members of the same subfamily had conservative gene structures and conserved motifs. The collinear analysis demonstrated that segmental duplication occurred widely in the TCP transcription factors of Ma bamboo, which mainly led to the expansion of a gene family. Cis-acting elements related to growth and development and stress response were found in the promoter regions of DlTCPs. Expression patterns revealed that DlTCPs have tissue expression specificity, which is usually highly expressed in shoots and leaves. Subcellular localization and transcriptional self-activation experiments demonstrated that the five candidate TCP proteins were typical self-activating nuclear-localized transcription factors. Additionally, the transcriptome analysis of the bamboo shoot buds at different developmental stages helped to clarify the underlying functions of the TCP members during the growth of bamboo shoots. DlTCP12-C, significantly downregulated as the bamboo shoots developed, was selected to further verify its molecular function in Arabidopsis. The DlTCP12-C overexpressing lines exhibited a marked reduction in the number of rosettes and branches compared with the wild type in Arabidopsis, suggesting that DlTCP12-C conservatively inhibits lateral bud outgrowth and branching in plants. This study provides useful insights into the evolutionary patterns and molecular functions of the TCP transcription factors in Ma bamboo and provides a valuable reference for further research on the regulatory mechanism of bamboo shoot development and lateral bud growth.

Project description:BackgroundBamboo occupies an important phylogenetic node in the grass family with remarkable sizes, woodiness and a striking life history. However, limited genetic research has focused on bamboo partially because of the lack of genomic resources. The advent of high-throughput sequencing technologies enables generation of genomic resources in a short time and at a minimal cost, and therefore provides a turning point for bamboo research. In the present study, we performed de novo transcriptome sequencing for the first time to produce a comprehensive dataset for the Ma bamboo (Dendrocalamus latiflorus Munro).ResultsThe Ma bamboo transcriptome was sequenced using the Illumina paired-end sequencing technology. We produced 15,138,726 reads and assembled them into 103,354 scaffolds. A total of 68,229 unigenes were identified, among which 46,087 were annotated in the NCBI non-redundant protein database and 28,165 were annotated in the Swiss-Prot database. Of these annotated unigenes, 11,921 and 10,147 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. We could map 45,649 unigenes onto 292 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. The annotated unigenes were compared against Moso bamboo, rice and millet. Unigenes that did not match any of those three sequence datasets are considered to be Ma bamboo unique. We predicted 105 unigenes encoding eight key enzymes involved in lignin biosynthesis. In addition, 621 simple sequence repeats (SSRs) were detected.ConclusionOur data provide the most comprehensive transcriptomic resource currently available for D. latiflorus Munro. Candidate genes potentially involved in growth and development were identified, and those predicted to be unique to Ma bamboo are expected to give a better insight on Ma bamboo gene diversity. Numerous SSRs characterized contributed to marker development. These data constitute a new valuable resource for genomic studies on D. latiflorus Munro and, more generally, bamboo.

Project description:Quantitative real-time PCR (qRT-PCR) has been emerged as an effective method to explore the gene function and regulatory mechanisms. However, selecting appropriate reference gene (s) is a prerequisite for obtaining accurate qRT-PCR results. Peach is one of important fruit in Rosaceae and is widely cultivated worldwide. In this study, to explore reliable reference gene (s) in peach with different types during fruit ripening and softening (S1-S4), nine candidate reference genes (EF-1α, GAPDH, TBP, UBC, eIF-4α, TUB-A, TUB-B, ACTIN, and HIS) were selected from the whole-genome data. Then, the expression levels of the nine selected genes were detected using qRT-PCR in three peach types, including 'Hakuho' (melting type), 'Xiacui' (stony hard type), 'Fantasia' and 'NJC108' (non-melting type) cultivars were detected using qRT-PCR. Four software (geNorm, NormFinder, BestKeeper and RefFinder) were applied to evaluate the expression stability of these candidate reference genes. Gene expression was characterized in different peach types during fruit ripening and softening stages. The overall performance of each candidate in all samples was evaluated. The Actin gene (ACTIN) was a suitable reference gene and displayed excellent stability in 'Total' set, 'Hakuho' samples, S3 and S4 fruit developmental stages. Ubiquitin C gene (UBC) showed the best stability in most independent samples, including 'Fantasia', 'NJC108', S2 sets. Elongation factor-1α gene (EF-1α) was the most unstable gene across the set of all samples, 'NJC108' and S2 sets, while showed the highest stability in 'Xiacui' samples. The stability of candidate reference genes was further verified by analyzing the relative expression level of ethylene synthase gene of Prunus persica (PpACS1) in fruit ripening and softening periods of 'Hakuho'. Taken together, the results from this study provide a basis for future research on the mining of important functional genes, expression patterns and regulatory mechanisms in peach.

Dataset Information

Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.

Background

Results

Conclusions

Publications

Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets