Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium

Accurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiata and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm, ΔCt, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage; PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen); EF1A and ArgK between two populations; β-tubulin and EF1A for different temperatures (5, 15, 25, 35 °C); and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.

Selection and Validation of Reference Genes for qRT-PCR in Lentinula edodes under Different Experimental Conditions

Lentinula edodes is the most consumed mushroom in Asia due to its nutritional and medicinal values, and the optimal reference gene is crucial for normalization of its gene expression analysis. Here, the expression stability of 18 candidate reference genes (CRGs) in L. edodes was analyzed by three statistical algorithms (geNorm, NormFinder and BestKeeper) under different stresses (heat, cadmium excess and Trichoderma atroviride infection), different substrates (straw, sawdust and corn stalk) and different development stages (mycelia, primordia and fruit bodies). Among the 18 CRGs, 28S, Actin and α-tub exhibited the highest expression stability in L. edodes under all conditions, while GPD, SPRYP and MSF showed the least stable expression. The best reference gene in different conditions was different. The pairwise variation values showed that two genes would be sufficient for accurate normalization under different conditions of L. edodes. This study will contribute to more accurate estimation of the gene relative expression levels under different conditions using the optimal reference gene in qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis.

Reference Gene Validation for Quantitative Real-time PCR Studies in Amphibian Kidney-derived A6 Epithelial Cells

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

Population structure and genetic diversity using microsatellite markers of four Algerian rabbit populations precludes hybridization with foreign breeds

The genetic characterization of our native rabbit populations is crucial for their development of genetic improvement and conservation programs. In the current study, fifteen microsatellite markers were used to investigate the genetic diversity and phylogenetic relationship among four Algerian popula-tions of rabbits; White (B), White and grey (G), Black and white (N) and Brown and white (M) in addition to Gabali (EG) and New Zealand White (EN) from Egypt. The microsatellites were INRACCDDV0003, SAT2, SAT3, SAT4, SAT5, SAT7, SAT8, SAT12, SAT13, SOL30, SOL33, SOL44, D3Utr2, D6Utr4 and D7Utr5. 90 animals were studied including 15 rabbits from each population. Results revealed that the average number of alleles per locus was 14.26 and the observed heterozygosity averaged 0.62 and ranging from 0.47 in marker SAT2 to 0.8 in marker, D6Utr4 while the expected heterozygosity averaged 0.72 and ranged from 0.62 in marker SAT2 to 0.77 in marker SOL44. The average polymorphic information content (PIC) was 0.85 and ranged from 0.77 at locus D3Utr2 to 0.93 at locus SOL33. Most loci showed deviations from Hardy-Weinberg Equilibrium with highly significant level. The inbreeding coefficient of the individuals relative to the total population (FIT) was the highest 0.28. The within-population heterozygote deficit (FIS) averaged 0.13. The pairwise variation among the populations (FST) averaged 0.16 and rang-ing from 0.09 for D3Utr2 to 0.27 for SAT2. The highest pairwise Nei’s genetic distance was recorded between G and EG (0.87) and the closest genetic dis-tance for natif population was between M and N (0.33). The smallest pair-wise FST was recorded between G and M (0.059). B, G and M, N populations were clustered together forming admixed mosaic cluster.