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• A systematic assessment of 18 potential reference genes (RGs) in 10 different ovine tissues was carried out to identify internal control genes for qPCR analysis. • Four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), were employed to rank the stability of these RGs. • RefFinder was used for a comprehensive ranking, identifying ACTB, PPIB, BACH1 , and B2M as the most stable RGs. • RPS9, RPS15 , and PGK1 showed variable expression across different tissues. • Validation was performed using qPCR analysis of four target genes in the skin from two sheep breeds. Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB , BACH1 , B2M , GAPDH , HMBS , HPRT1 , PGK1 , PPIA , PPIB , RPLP0 , RPL19 , RPS9 , RPS15 , RPS28 , SDHA , TBP , UXT , and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1 , and B2M emerged as the most stable RGs, while RPS9, RPS15 , and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1 , and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1 , and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues. [ABSTRACT FROM AUTHOR]

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L . japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L . japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L . japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT , 18S rRNA , and RPL13 during different stages; AK , RPL13 , and TBP among sexes; EF1A , PPI , and RPL27 in different tissues, and EF1A , RPL13 , and PPI in adults fed on different diets. Moreover, the expression profile of a target gene ( odorant receptor 1 , OR1 ) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L . japonica and will benefit subsequent genomics and functional genomics research on this natural enemy. [ABSTRACT FROM AUTHOR]

The increasing demand of strawberry ( Fragaria × ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase ( PAL ) and 9-cis epoxycarotenoid dioxygenase ( NCED1 ) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts. [ABSTRACT FROM AUTHOR]