Identification and evaluation of suitable reference genes for gene expression analysis in rubber tree leaf

Abstract Backgroud Gene expression profiling is increasingly applied to investigate molecular mechanisms for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber, however, little is known about reference genes in leaf. Results The main objective of this current study was to identify some reference genes with stable expression patterns in leaf at various developmental stages, as well as during abiotic (temperature extremes) and biotic stresses. Gene expression profiling experiments in rubber tree leaf identified the ubiquitin-proteasome system as having excellent potential as reference genes. Among a total of 30 tested genes investigated, 24 new (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new genes had better stability of expression when compared with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes in rubber tree leaf in four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data. Conclusion Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other species.

Download Full-text

Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

PeerJ ◽

10.7717/peerj.2428 ◽

2016 ◽

Vol 4 ◽

pp. e2428 ◽

Cited By ~ 5

Author(s):

Guanglin Niu ◽

Yalan Yang ◽

YuanYuan Zhang ◽

Chaoju Hua ◽

Zishuai Wang ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Muscle Development ◽

Expression Stability ◽

Skeletal Muscle Development ◽

Expression Levels ◽

Suitable Reference

The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes (ACTB,GAPDH,RNF7,RHOA,RPS18,RPL32,PPIA,H3F3,API5,B2M,AP1S1,DRAP1,TBP,WSB, andVAPB) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated thatGAPDHandACTBhad the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development.RPS18,API5, andVAPBhad stable expression levels in prenatal stages, whereasAPI5,RPS18,RPL32, andH3F3had stable expression levels in postnatal stages.API5andH3F3expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.

Download Full-text