Reference Genes Research Articles

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis in Cockroach Parasitoid Tetrastichus hagenowii (Ratzeburg).

Parasitoid wasps play a crucial role in the efficient control of pests, a substantial menace to human health and well-being. Tetrastichus hagenowii (Ratzeburg) stands out as the most effective egg parasitoid wasp for controlling American cockroaches, but accurate and stable reference genes for quantitative real-time polymerase chain reaction of T. hagenowii genes are still lacking. In this study, we assessed seven candidate nuclear genes, including α-tubulin (α-TUB), elongation factor-1-alpha (EF-1α), β-actin (Actin), ribosomal protein 49 (RP49), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nicotinamide adenine dinucleotide (NADH), and elongation factor 2 (EF2) of T. hagenowii. By analyzing expression stability with four algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), as well as comprehensive ranking with RefFinder, we identified α-TUB as the most stable reference gene for the larval, pupal, female adult, and male adult stages. Subsequently, we estimated the transcript levels of vitellogenin (Vg) and cuticle protein (CP) after normalization with α-TUB across various developmental stages. Significantly higher expression levels of CP and Vg were observed in pupae and female adults, respectively, consistent with previous findings in other insects. This study offers a reliable reference gene for normalizing transcription levels of T. hagenowii genes.

Molecular analysis of the reactions in Salicornia europaea to varying NaCl concentrations at various stages of development to better exploit its potential as a new crop plant.

Freshwater scarcity demands exploration of alternative resources like saline water and soils. Understanding the molecular mechanisms behind NaCl regulation in potential crop plants becomes increasingly important for promoting saline agriculture. This study investigated the euhalophyte Salicornia europaea, analyzing its gene expression, yield, and total phenolic compounds under hydroponic cultivation. We employed five salinity levels (0, 7.5, 15, 22.5, and 30 g/L NaCl) across five harvests at 15-day intervals, capturing plant development. Notably, this design deviated from conventional gene expression studies by recording organ-specific responses (shoots and roots) in plants adapted to long-term salinity treatments at various developmental stages. The highest fresh mass of S. europaea was observed four months after germination in 15 g/L NaCl. Identifying a reliable set of reference genes for normalizing gene expression data was crucial due to comparisons across shoots, roots, developmental stages, and salinity levels. A set of housekeeping genes - ubiquitin c (SeUBC), actin (SeActin) and dnaJ-like protein (SeDNAJ) - was identified for this purpose. Interestingly, plants grown without NaCl (0 g/L) displayed upregulation of certain genes associated with a NaCl deficiency related nutritional deprivation. These genes encode a tonoplast Na+/H+-antiporter (SeNHX1), a vacuolar H+-ATPase (SeVHA-A), two H+-PPases (SeVP1, SeVP2), a hkt1-like transporter (SeHKT), a vinorine synthase (SeVinS), a peroxidase (SePerox), and a plasma membrane Na+/H+-antiporter (SeSOS1). Other genes encoding an amino acid permease (SeAAP) and a proline transporter (SeProT) demonstrated marginal or dispersing salinity influence, suggesting their nuanced regulation during plants development. Notably, osmoregulatory genes (SeOsmP, SeProT) were upregulated in mature plants, highlighting their role in salinity adaptation. This study reveals distinct regulatory mechanisms in S. europaea for coping with varying salinity levels. Identifying and understanding physiological reactions and sodium responsive key genes further elucidate the relationship between sodium tolerance and the obligate sodium requirement as a nutrient in euhalophytes.

Selection of Reference Genes in Siraitia siamensis and Expression Patterns of Genes Involved in Mogrosides Biosynthesis.

Siraitia siamensis is a traditional Chinese medicinal herb. In this study, using S. siamensis cultivated in vitro, twelve candidate reference genes under various treatments were analyzed for their expression stability by using algorithms such as GeNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. The selected reference genes were then used to characterize the gene expression of cucurbitadienol synthase, which is a rate-limiting enzyme for mogroside biosynthesis. The results showed that CDC6 and NCBP2 expression was the most stable across all treatments and are the best reference genes under the tested conditions. Utilizing the validated reference genes, we analyzed the expression profiles of genes related to the synthesis pathway of mogroside in S. siamensis in response to a range of abiotic stresses. The findings of this study provide clear standards for gene expression normalization in Siraitia plants and exploring the rationale behind differential gene expression related to mogroside synthesis pathways.

PPIA, HRPT1, and PGK1 genes as the appropriate combination for RT-qPCR normalization in alveolar and femoral bone remodeling in olanzapine-treated rats.

Reliable gene expression analysis in bone remodeling studies requires an appropriate selection of internal controls, i.e. stable reference genes for the normalization of quantitative real-time PCR (RT-qPCR), the most common method used for quantifying gene expression measurements. Even the most widely used reference genes can have variable expression under different experimental conditions, or in different tissue types or treatment regimes, so selecting appropriate controls is a key step in ensuring reliable results. The aim of this research was to identify the most stable reference gene(s) for the study of olanzapine modulated bone remodeling in rats. RNA was isolated from the maxillary alveolar and femoral bones of olanzapine or placebo-treated Wistar rats and transcribed to cDNA. The expression of 12 candidate reference genes was assessed by RT-qPCR. Their expressions were analysed using GeNorm, NormFinder, BestKeeper and delta Ct algorithms, and by the comprehensive ranking method. PPIA, HRPT1 and PGK1 were the most stably expres sed reference genes and the combination of the three genes was optimal for normalization. This study is the first to identify the optimal reference genes for research in olanzapine-exposed rats, which serve as a pivotal benchmark for enhancing the accuracy and reliability of future RT-qPCR expression in bone studies.

BCORL1, POF1B, and USP9X copy number variation in women withidiopathic diminished ovarian reserve.

To analyze the copy number variation (CNV) in the X-linked genes BCORL1, POF1B, and USP9X in idiopathic diminished ovarian reserve (DOR). This case-control study included 47 women, 26 with DOR and 21 in the control group. Age, weight, height, BMI, and FSH level were evaluated, as well as antral follicle count (AFC), oocyte retrieval after controlled ovarian stimulation, and metaphase II (MII) oocytes. The CNVs of BCORL1, USP9X, and POF1B genes were measured by quantitative real time PCR (qPCR) using two reference genes, the HPRT1 (X-linked) and MFN2 (autosomal). Protein-protein interaction network and functional enrichment analysis were performed using the STRING database. The mean age was 36.52 ± 4.75 in DOR women and 35.38 ± 4.14 in control. Anthropometric measures did not differ between the DOR and control groups. DOR women presented higher FSH (p = 0.0025) and lower AFC (p < .0001), oocyte retrieval after COS (p = 0.0004), and MII oocytes (p < .0001) when compared to the control group. BCORL1 and POF1B did not differ in copy number between DOR and control. However, DOR women had more copies of USP9X than the control group (p = 0.028). The increase in the number of copies of the USP9X gene may lead to overexpression in idiopathic DOR and contribute to altered folliculogenesis and oocyte retrieval.

Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses.

Holotrichia parallela is among the world's most destructive pests. For accurate qPCR and gene expression studies, the selection of stable and appropriate reference genes is crucial. However, a thorough evaluation of potential reference genes for use in H. parallela research is lacking. In this study, 11 reference genes (GAPDH, RPL32, RPL7A, RPS18, RPL13a, RPL18, Actin, RPS7, RPS3, VATB,and EF1A) were evaluated under different biological conditions and environmental stresses. The stability of 11 potential reference gene transcripts was evaluated through various computational tools, including geNorm, BestKeeper, NormFinder, theΔCt method, and the RefFinder program. Under various developmental stages and RNAi conditions, RPL18 and RPL13a exhibited the greatest stability. RPL13a, RPL18, and RPL32 were the most stable genes in both male and female adults. Under differing tissue conditions, RPL13a and RPS3 stood out as the most reliable. Moreover, under varying photoperiod conditions, RPL13a, RPS3 and RPL32 were the most stable genes. Lastly, Actin and RPL13a were the most stable genes across different temperatures. These findings offer essential criteria for selecting suitable reference genes across diverse experimental settings, thereby establishing a solid basis for accurate gene expression studies in H. parallela using RT-qPCR.

MiR-122, miR-133a, and miR-206 as potential biomarkers for post-mortem interval estimation.

Accurate estimation of post-mortem interval (PMI) is crucial in forensic investigations. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that remain relatively stable within the cell nucleus despite post-mortem changes. We assessed three target genes (miR-122, miR-133a, and miR-206) for PMI estimation using 72 healthy adult male BALB/c mice exposed to two different temperatures (4 and 21℃) at nine different time points over 10days. Initially, the stability of the two reference genes (RNU6B and 5 srRNA) was evaluated using gene stability analysis tools (Delta Ct, Best Keeper, and Genorm) to select the optimal reference gene. RNU6B was found to be the most stable endogenous control. Subsequently, the expression patterns of miR-122, miR-133a, and miR-206 were analyzed within a 10-day PMI period using the heart, skeletal muscle, liver, and brain tissues. At 4℃, miR-122 levels significantly decreased on days 8 and 10 in all tissues, with only the liver showing significant changes at 21℃. MiR-133a decreased over time in the heart, muscles, and brain, showing a dramatic decrease on days 8 and 10 in the heart and muscles at both temperatures. Although miR-206 levels decreased over time in muscles and liver at 4℃, these increased in the brain at 21℃, with no expression changes in other organs. In summary, miR-122, miR-133a, and miR-206 are potential PMI markers in heart and skeletal muscle tissues.

Selection and Validation of Reliable Reference Genes for Liquidambar formosana Leaves with Different Leaf Colors.

Liquidambar formosana Hance is renowned for its rich leaf color and possesses notable advantages, such as robust adaptability, strong resistance to diseases and pests, and rapid growth, making it a preferred choice for urban greening and carbon sequestration forest initiatives. The completion of whole-genome sequencing of L. formosana has spurred an increased interest in exploring the molecular mechanisms underlying seasonal changes in leaf color, marking a significant focus in L. formosana breeding research. However, there is currently a lack of stable reference genes suitable for analyzing the expression patterns of functional genes in L. formosana exhibiting varying leaf colors. This study selected five L. formosana varieties with significant differences in leaf colors. Through the RT-qPCR analysis, and evaluation using BestKeeper, geNorm, NormFinder, Delta Ct, and RefFinder, the expression stability of 14 candidate reference genes was examined. Consequently, two reference genes (LifEF1-α and LifACT) with stable expression, suitable for RT-qPCR of L. formosana with diverse leaf colors, were identified. The stability of these selected reference genes was further validated by examining the LifbHLH137 gene, which promoted the biosynthesis of anthocyanins. This advancement facilitated molecular biology and genetic breeding investigations of L. formosana, providing essential data for the precise quantification of functional genes associated with leaf color variation.

Transcriptome-level analysis of gene expressions in different tissues of tomato and key gene identifications during seed germination

Exploring the spatiotemporal expression patterns of genes is the foundation for studying their functions and molecular mechanisms. Genes with stable spatiotemporal expression patterns are often used as reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) and genes are significantly highly expressed in a specific tissue or stage often represent their unique biological functions. Tomato is an important economic crop and a model plant for scientific researches. However, the existing reference genes in tomatoes are not stable enough and researches on tissue-specific genes have been more focused on flowers and fruits, with less emphasis on studying the seed germination process. In this study, a total of 26 tissue/organ samples from the entire growth cycle of tomatoes were collected from Micro-Tom (Solanum lycopersicum cv. Micro-Tom). After RNA sequencing, a transcriptome database of the entire growth cycle of tomatoes was constructed. Solyc01g107870.3 gene was identified as a novel reference gene through systematic analysis and validated by qRT-PCR. Moreover, tissue-specific genes in seed and 8 days past germination (DPG) seedlings are screened. Among them, SlLEA16 gene belongs to late embryogenesis abundant protein (LEA) gene family, having subcellular localization in the nucleus and cytoplasm, and playing important roles in seed germination initiation. The above experimental results provide data support for studying tomato gene spatiotemporal expression patterns and can also serve as an experimental basis for investigating the deeper mechanisms of tomato seed germination processes.

Evaluation of suitable reference genes for qPCR normalisation of gene expression in a Achilles tendon injury model.

Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.

Evaluation of Reference Gene Stability in Goat Skeletal Muscle Satellite Cells during Proliferation and Differentiation Phases.

The process of skeletal muscle development is intricate and involves the regulation of a diverse array of genes. Accurate gene expression profiles are crucial for studying muscle development, making it essential to choose the right reference genes for real-time quantitative PCR (RT-qPCR). In the present study, eight candidate reference genes were identified from our previous transcriptome sequencing analysis of caprine skeletal muscle satellite cells (MuSCs), and two traditional reference genes (ACTB and GAPDH) were assessed. The quantitative levels of the candidate reference genes were determined through the RT-qPCR technique, while the stability of their expression was evaluated utilizing the GeNorm, NormFinder, BestKeeper, and RefFinder programs. Furthermore, the chosen reference genes were utilized for the normalization of the gene expression levels of PCNA and Myf5. It was determined that conventional reference genes, including ACTB and GAPDH, were not appropriate for normalizing target gene expression. Conversely, RPL14 and RPS15A, identified through RNA sequencing analysis, exhibited minimal variability and were identified as the optimal reference genes for normalizing gene expression during the proliferation and differentiation of goat MuSCs. Our research offers a validated panel of optimal reference genes for the detection of differentially expressed genes in goat muscle satellite cells using RT-qPCR.

Identification of reference genes via real-time quantitative PCR for investigation of the transcriptomic basis of the squalene biosynthesis in different tissues on olives under drought stress

Squalene plays a crucial role in plant growth, development, and stress tolerance. To select the best reference genes (RGs) for expression profile analysis of genes involved in squalene biosynthesis in olives under stress tolerance, the expression stability of 22 candidate RGs across four tissues (root, stem, leaf, and fruit) and five representative cultivars under drought stress was assessed using six methods. Our study showed that, ubiquitin-conjugating enzyme1 (UBC1) and 60S ribosomal protein L18-2-like (60S) were the most stable RGs across five different cultivars and various tissues. Additionally, elongation factor 1-alpha-like (EF1-α2) and UBC1 were the most stable RG in olives under drought stress. UBC1 was the appropriate RG for further study, qPCR analysis showed that five certain genes involved in the squalene biosynthesis exhibited significant differential expression under varying conditions. Specially, the expression level of squalene synthase (SQS) in the leaves was highest, and HPLC analysis showed that squalene content was the highest in leaves. Likewise, the expression levels of SQS and farnesyl diphosphate synthase (FPPS) in the leaves of the cultivar Arbosana and Chelmsford Lal were significantly higher than those of the other cultivars, respectively, and HPLC analysis also showed that squalene content was highest in the two cultivars. Interestingly, the amount of squalene increased dramatically in olive under drought stress, as lupeol synthase (LUPS) was significantly up-regulated. To date, this study firstly provided the comprehensive analysis of RGs related to squalene biosynthesis in various cultivars and tissues of olives under drought stress.

Identification and validation of reference genes for RT-qPCR analysis in Iris domestica under Cd stress

Iris domestica is a widely used ornamental garden and important medicinal plant. Our previous studies have shown that it exhibits significant uptake and translocation capacity under Cd stress compared to other Iris species. Gene expression is studied using RT-qPCR; however, there are no reference genes have been found for I. domestica under Cd stress. In this investigation, thirteen possible reference genes from previous studies and our transcriptome were screened using RT-qPCR in the leaves and roots of Cd-stressed plants. The findings revealed that UBC9 and ACT were the best reference genes for roots with and without Cd stress, whereas YLS8 and ACT7 were the best reference genes for leaves. Among the different tissues without Cd stress, UBC9 and UBC28 exhibited the best results, whereas PP2C06 and UBC9 exhibited the best results under Cd stress. The most stable reference genes in the leaves and roots were UBC9 and UBC28, respectively, under and without Cd stress, and GADPH was the most unstable. Finally, three metal ion response genes, NRAMP2, YSL9 and CYP81Q32 were detected using RT-qPCR and compared with the transcriptome data to further confirm the reliability of the chosen genes. This study identified suitable reference genes for I. domestica under Cd-stress conditions.

Selection and Validation of Reference Genes in Dendrocalamus brandisii for Quantitative Real-Time PCR.

Dendrocalamus brandisii (Munro) Kurz is a sympodial bamboo species with a wide distribution in tropical and subtropical regions. Due to its remarkable regenerative ability and exceptional flavor, this species plays a pivotal role in bolstering the economies of numerous nations across these regions. We recently published a high-quality genome of this species. To date, no study results have identified the optimal reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) normalization in Dendrocalamus brandisii. qRT-PCR offers a highly accurate and effective approach to analyzing gene expression. However, the precision of the resulting quantitative data hinges on the correct choice of reference genes. Twenty-one potential reference genes were identified from the D. brandisii transcriptomes. Their expression in 23 samples, including 8 different tissue organs and 15 samples of D. brandisii under various treatment conditions, were evaluated through qRT-PCR. Subsequently, four software programs-Delta CT, geNorm, NormFinder, and RefFinder-were employed to compare their expression stability. The results revealed that the selection of optimal reference genes varied based on the particular organ and condition being examined. EF-1-α-2 consistently exhibits the most stable expression across diverse tissues, while ACTIN-1, TUBULIN-1, and EF-1-α-2 were the most consistent reference genes in roots, culms, and leaves under various treatments, respectively. In this study, we identified and characterized appropriate internal genes utilized for calibrating qRT-PCR analyses of D. brandisii across different tissue organs and under various treatments. This research will provide key insights for advancing the study of gene functionality and molecular biology in D. brandisii and related species.

An experimental study to estimate the early postmortem interval based on the degradation of lncRNAs in rat brain tissue

To study the degradation of lncRNAs in EPMI in rat brain tissue, this study provides a new direction for the estimation of EPMI. LncRNA high-throughput sequencing was performed on the brain tissues of hemorrhagic shock model rats at 0 h and 24 h, and the target lncRNAs were screened. Samples at 0, 1, 3, 6, 12, 18 and 24 h after death were collected, and miRNA-9 and miRNA-125b were used as reference genes. The relative expression levels of lncRNAs at each PMI were detected by RT–qPCR, and a functional model involving lncRNAs and EPMI was established. Samples were collected at 6, 9, 15, and 21 h after death for functional model verification. The expression of several lncRNAs decreased with the prolongation of EPMI, and the mathematical model established by several lncRNA indices exhibited good fit. The verification results of the multi-index joint function model are significantly better than those of the single-index function model, and the established model is more practical. There is a linear relationship between lncRNAs and EPMI, and the multi-index function model is significantly better than the single-index function model, which is important for EPMI inference in forensic pathology practice.

Increased expression of ER stress, inflammasome activation, and mitochondrial biogenesis-related genes in peripheral blood mononuclear cells in major depressive disorder.

A subset of major depressive disorder (MDD) is characterized by immune system dysfunction, but the intracellular origin of these immune changes remains unclear. Here we tested the hypothesis that abnormalities in endoplasmic reticulum (ER) stress, inflammasome activity and mitochondrial biogenesis contribute to the development of systemic inflammation in MDD. RT-qPCR was used to measure mRNA expression of key organellar genes from peripheral blood mononuclear cells (PBMCs) isolated from 186 MDD and 67 healthy control (HC) subjects. The comparative CT (2-ΔΔCT) method was applied to quantify mRNA expression using GAPDH as the reference gene. After controlling for age, sex, BMI, and medication status using linear regression models, expression of the inflammasome (NLRC4 and NLRP3) and the ER stress (XBP1u, XBP1s, and ATF4) genes was found to be significantly increased in the MDD versus the HC group. Sensitivity analyses excluding covariates yielded similar results. After excluding outliers, expression of the inflammasome genes was no longer statistically significant but expression of the ER stress genes (XBP1u, XBP1s, and ATF4) remained significant and the mitochondrial biogenesis gene, MFN2, was significantly increased in the MDD group. NLRC4 and MFN2 were positively correlated with serum C-reactive protein concentrations, while ASC trended significant. The altered expression of inflammasome activation, ER stress, and mitochondrial biogenesis pathway components suggest that dysfunction of these organelles may play a role in the pathogenesis of MDD.

A portable multifunctional biosensor based on a newly screened endogenous reference gene for pork adulteration detection

The adulteration of animal-derived food is a widespread problem with meat adulteration emerging as a global concern. To address the need for on-site rapid detection and visual analysis, a portable multifunction biosensor (PMB) was developed. This device integrates Recombinase Polymerase Amplification (RPA), CRISPR/Cas12a assay, and a custom-designed 3D-printed multifunction device, specifically for the visual and rapid detection of pork in food. Initially, a screening model was constructed using Perl bioinformatics based on chromosomal gene information, and the LOC110262058 gene was selected as a reliable endogenous reference gene for pigs. Subsequently, a one-tube RPA-CRISPR/Cas12a system was established to simplify the procedures, converting pork-specific signals into fluorescence while preventing cross-contamination. In addition, an independently designed 3D-printed multifunction device was developed to support the RPA-CRISPR/Cas12a system for one-tube detection, incorporating functions such as heating, centrifugation, and imaging. The reaction conditions of the PMB were optimized to reduce the reaction time to 20–30 min with a sensitivity of 0.01 g/100 g. The analysis of actual samples indicated that the PMB has high selectivity and practical effectiveness for pork detection. The proposed biosensor offers several detection advantages, including output, ease of use, stability, and independence from complex equipment, making it a powerful method for rapid pork detection in food.

Evaluation of reference genes suitable for studying mRNAs and microRNAs expression in thyroid neoplasms

Analysis of gene expression is a pivotal method at the core of biomarkers studies and cancer research. Currently, RT-qPCR is the most commonly used technique to investigate the expression of certain genes. The accurate and reliable result relies on an effective normalization step using suitable reference genes. The present study was designed to evaluate the eligibility of a set of candidate mRNAs and snoRNA as reference genes in the most common human thyroid neoplasms. We tested the expression levels of eleven mRNA and small RNA housekeeping genes in thyroid samples. The stability of the candidate genes was examined in different thyroid lesions and under different experimental conditions. Results were compared to the reported data in the TCGA database. Our results suggested HPRT1 and ACTB as the best mRNA reference genes, SNORD96A, and SNORD95 as the best miRNA reference genes in thyroid tissues. These genes showed the most stable expression pattern among different thyroid lesions as well as different experimental conditions. The findings in this study highlight the effect of reference genes selection on data interpretation and emphasize the importance of testing for suitable reference genes to be used in specific types of cells and experimental conditions to ensure the validity and accuracy of results.

CYP2D6 copy number determination using digital PCR.

CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the "One-pot" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5'UTR, intron 6, and exon 9 regions. Genomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA. Overall, results between the two platforms and digestions methods were consistent. The "One-pot" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference. The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

Efficient CRISPR/Cas9-mediated ebony gene editing in the greater wax moth Galleria mellonella.

The greater wax moth, Galleria mellonella (Lepidoptera, Pyralidae), is a major bee pest that inflicts considerable harm on beehives, leading to economic losses. It also serves as a valuable resource insect and a model organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system plays a crucial role in improving economic insect breeding and developing efficient agricultural pest management systems in Lepidoptera. However, the CRISPR/Cas9 protocols have not been developed for G. mellonella. Here, the Gmebony knockout (KO) strain was established using the CRISPR/Cas9 genome editing system. We obtained Gmebony KO strain in the G4 generation, which took approximately 10 months. When compared with wild-type, the head, notum, and the terminal abdominal surface of 1st to 4th instar larvae in the KO strain changed from yellow to brown, and these regions of the KO strain gradually transformed into a black color from the 5th instar larvae, and the body color of the adult moth in the KO strain changed to black. The developmental period of the early larval and the following larval instars extended. The embryonic hatchability of the Gmebony KO strain was significantly decreased. The pupal body weight of the Gmebony KO strain was not affected. The feasibility of the CRISPR/Cas9 methodology was validated by single-target editing of Gmebony. Our findings provide the first evidence that the ebony gene can serve as a pigmentation reference gene for genetic modifications of G. mellonella. Meanwhile, it can be utilized in the development of genome editing control strategies and for gene function analyses in G. mellonella.