Stable Reference Genes Research Articles

Evaluation of suitable reference genes for expression profile analyses of target genes in the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae).

The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae), is a major destructive insect pest of coffee, which impacts the coffee crops negatively. As a draft genome has been completed for this insect, most molecular studies on gene transcriptional levels under different experimental conditions will be conducted using real-time reverse-transcription quantitative polymerase chain reactions (RT-qPCR). However, the lack of suitable internal reference genes will affect the accuracy of RT-qPCR results. In this study, the expression stability of nine candidate reference genes was evaluated under different developmental stages, temperature stress, and Beauveria bassiana infection. Data analyses were completed by four commonly used programs, BestKeeper, NormFinder, geNorm, and RefFinder. The result showed that RPL3 and EF1α combination were recommended as the most stable reference genes for developmental stages. EF1α and RPS3a combination were the top two stable reference genes for B. bassiana infection. RPS3a and RPL3 combination performed as the optimal reference genes both in temperature stress and all samples. Our results should provide a good foundation for the expression profile analyses of target genes in the future, especially for molecular studies on insect genetic development, temperature adaptability, and immune mechanism to entomogenous fungi in H. hampei.

Selection of reference genes in liproxstatin-1-treated K562 Leukemia cells via RT-qPCR and RNA sequencing.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) can accurately detect relative gene expression levels in biological samples. However, widely used reference genes exhibit unstable expression under certain conditions. Here, we compared the expression stability of eight reference genes (RPLP0, RPS18, RPL13, EEF1A1, β-actin, GAPDH, HPRT1, and TUBB) commonly used in liproxstatin-1 (Lip-1)-treated K562 cells using RNA-sequencing and RT-qPCR. The expression of EEF1A1, ACTB, GAPDH, HPRT1, and TUBB was considerably lower in cells treated with 20 μM Lip-1 than in the control, and GAPDH also showed significant downregulation in the 10 μM Lip-1 group. Meanwhile, when we used geNorm, NormFinder, and BestKeeper to compare expression stability, we found that GAPDH and HPRT1 were the most unstable reference genes among all those tested. Stability analysis yielded very similar results when geNorm or BestKeeper was used but not when NormFinder was used. Specifically, geNorm and BestKeeper identified RPL13 and RPLP0 as the most stable genes under 20 μM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable under 10 μM Lip-1 treatment. TUBB and EEF1A1 were the most stable genes in both treatment groups according to the results obtained using NormFinder. An assumed most stable gene was incorporated into each software to validate the accuracy. The results suggest that NormFinder is not an appropriate algorithm for this study. Stable reference genes were recognized using geNorm and BestKeeper but not NormFinder. Overall, RPL13 and RPLP0 were the most stable reference genes under 20 μM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable genes under 10 μM Lip-1 treatment.

Identification of stable reference genes for relative quantification of long RNA expression in urinary extracellular vesicles

AbstractUrinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA‐seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA‐seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1‐2, RPL4, GAPDH, RPS27A as candidate reference genes. RT‐qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.

Identification of specific reference gene for normalization of RT-qPCR data in rhythmic gene expression studies of the effect of developmental hormone antagonist in postembryonic development in Bombyx mori.

Bombyx mori is a lepidopteran holometabolous insect with distinct developmental stages: egg, larvae, pupae, and adult. The lepidopteran insect undergoes major modifications in the central nervous system (CNS) so as to adapt to the lifestyle of these distinct stages with specific habitats and functions from voraciously feeding larval stages to flying reproductive adults via dormant pupal stages. Such transitions are linked to transcriptional, epigenetic, and translational complexities. Therefore, studying rhythmic gene expression in CNS of various developmental stages and the effects of antagonists on developmental hormones requires a very stable reference gene (RG). To facilitate rhythmic gene expression studies using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in B. mori and the effect of developmental hormone juvenile hormone (JH) and 20-hydroxy ecdysone hormone (20 HE), antagonists Precocene 1 and testosterone, respectively, were used. Eight candidate RGs, namely, Translational initiation factor 3 subunit 4 (TI3S4), Translational initiation factor 3 subunit 5 (TI3S5), Ribosomal protein subunit 7 (RPs7), TATA-binding protein association factor (TAF13), Translational initiation factor 4 A (TI4A), Ribosomal protein (RPL32), Elongation factor 1 (EF1), and Arginine kinase (AK), were assessed in the CNS of B. mori. The postembryonic developmental (PED) stages used were the fifth late larval instar, early pupa, mid pupa, late pupa, and adult. The assessments were done at four different time points, Zeitgeber time (ZT) 0, 6, 12, and 18, to find stability towards 24-h rhythmic expression. RefFinder, geNorm, and Ct value analysis were performed. RefFinder and geNORM studies suggested stability order as TI3S4 > TI3S5 > RPs7, but Ct value evaluation showed stability order as TI3S5 > TI3S4 > RPs7. We therefore demonstrated that TI3S4, TI3S5, and RPs7 can be used as RG in various PED stages in CNS of B. mori (Strain: CB-hybrid, PM×CSR2) towards studies with effects of JH and 20 HE antagonists.

Standardization of reference genes for real-time PCR analysis of Vigna radiata L. under Agrobacterium tumefaciens infection

Gene expression analysis using real-time polymerase chain reaction (PCR) requires the validation of a stable reference gene for normalization. Previously reported candidates may not be suitable for novel crops. Vigna radiata is a protein-rich legume crop used in many countries across the world. As this crop is recalcitrant to transformation such as most legumes, we were interested in V. radiata-Agrobacterium interaction studies. Toward this end, we wanted to identify a suitable reference for normalization under Agrobacterium-infections conditions. We selected seven candidate genes (Actin, Tubulin, Glyceraldehyde 3-phosphate dehydrogenase, Ubiquitin 10, Cyclophilin, 18S Ribosomal RNA, and F-box protein) and performed temporal-expression analysis using real-time PCR at 3 h, 24 h, and 4-day post-infection. When their Ct values were compared using the routinely used four algorithms, Normfinder, geNorm, Bestkeeper, and RefFinder, each suggested a different candidate gene. We narrowed-down to Cyclophilin by taking the statistical approach of using analysis of variance and Tukey honestly significant difference. Thus, our report signifies the use of additional statistical analyses in case of situations of discrepancies due to variable suggestions by software.

From stability to reliability: Unveiling the un-biased reference genes in porcine ovarian granulosa cells under different conditions

Selection of optimal reference genes (RGs) is fundamental for functional genomics studies and gene expression analysis, which are two main approaches to identify functional genes and their expression patterns. However, no systematic study has identified the suitable RGs in porcine ovarian granulosa cells (GCs) which are essential for follicle fate and sow fertility. In this study, the expression profiles of 12 widely-used RGs (GAPDH, RPLP0, ACTB, TUBA1B, EIF3K, PPIA, ATP5F1, B2M, HPRT1, UBC, RPS3, and EEF1A1) in porcine GCs during follicular development and under different abiotic stresses were systematically investigated. Expression stability of the candidate RGs were comprehensively accessed by five statistical algorithms including ΔCt, NormFinder, BestKeeper, geNorm, and RefFinder, indicating that RPS3 and PPIA are the optimal RGs during follicular development, EEF1A1 and RPLP0 are most stable under oxidative stress and inflammation, while ATP5F1, B2M, and RPS3 have higher stability under starvation and heat stress. Notably, the most commonly used RGs (ACTB, GAPDH, and TUBA1B) exhibited low stability in GCs. Reliability of stable RGs was verified by RT-qPCR and showed that selection of the stable RGs significantly improved the detection accuracy of qPCR, which confirms once again that the stability of RGs should not be taken for granted. Our findings identified optimal RG sets in porcine GCs under different conditions, which is helpful in future studies to accurately identify the key regulators and their expression patterns during follicular development in sows.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae).

Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data normalization strongly depends upon stable reference genes. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (RPL32, RPS23, SDHA, UBQ, UCCR, GST, TATA-box, HSP70, GAPDH, RPL7A, SOD, RPS3, Actin, α-tubulin, AK, and EF1) were carefully chosen in R. pedestris, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in R. pedestris is contingent upon the specific experimental conditions. RPL7A and EF1 are optimal reference genes for developmental stages. Furthermore, α-tubulin and EF1 exhibit the most stable expression across various adult tissues. RPL32 and RPL7A exhibit the most stable expression for adult age. For nymph age, RPL32 and SOD display the most stable expression. For temperature conditions, RPS23 and RPL7A were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (RpedOBP37) and cytochrome P450 6a2 (RpedCYP6) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on R. pedestris, serving as a fundamental groundwork for future investigations on stable reference genes in R. pedestris as well as other organisms.

Transcriptome-Based Identification of the Optimal Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Analyses of Lingonberry Fruits throughout the Growth Cycle.

(1) Background: Vaccinium vitis-idaea is a nutritionally and economically valuable natural wild plant species that produces berries useful for treating various diseases. There is growing interest in lingonberry, but there is limited information regarding lingonberry reference genes suitable for gene expression analyses of different tissues under various abiotic stress conditions. The objective of this study was to identify stable reference genes suitable for different lingonberry tissues in response to abiotic stress. (2) Methods: The delta Ct method and the GeNorm v3.5 and NormFinder v20 programs were used to comprehensively analyze gene expression stability. (3) Results: Actin Unigene23839 was the best reference gene for analyzing different cultivars, whereas Actin CL5740.Contig2 was the most suitable reference gene for analyzing different tissues and alkali stress. In contrast, 18S rRNA CL5051.Contig1 was the most stable reference gene under drought conditions. (4) Conclusions: These suitable reference genes may be used in future qRT-PCR analyses of different lingonberry tissues and the effects of abiotic stresses. Furthermore, the study data may be useful for functional genomics studies and the molecular breeding of lingonberry. In summary, internal reference genes or internal reference gene combinations should be carefully selected according to the experimental conditions to ensure that the generated gene expression data are accurate.

Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Colletotrichum fructicola from Litchi.

Litchi (Litchi chinensis Sonn.) is a tropical fruit originating from southern China that is currently cultivated in subtropical and tropical regions worldwide. Litchi anthracnose, caused by Colletotrichum fructicola, a dominant species of Colletotrichum spp., is an important disease of litchi that damages the fruits in fields and in post-harvest storage. Real-time quantitative PCR (RT-qPCR) is a common technique with which to detect the expression of and function of target genes quickly and precisely, and stable reference genes are crucial. However, there is no comprehensive information on suitable reference genes of C. fructicola present. Here, we designed eight candidate genes (GAPDH, α-tubulin, 18S, β-tubulin, EF1a, TATA, RPS5, and EF3) using RefFinder software (programs: geNorm, ΔCt, BestKeeper, and NormFinder) to investigate their reliability in the detection of C. fructicola under five different treatments (fungal development stage, temperature, UV, culture medium, and fungicide). The results showed the optimal reference genes under different conditions: EF1a and α-tubulin for developmental stage; α-tubulin and β-tubulin for temperature; α-tubulin and RPS5 for UV treatment; RPS5 and α-tubulin for culture medium; α-tubulin, GAPDH, and TATA for fungicide treatments. The corresponding expression patterns of HSP70 (Heat shock protein 70) were significantly different when the most and the least stable reference genes were selected when treated under different conditions. Our study provides the first detailed list of optimal reference genes for the analysis of gene expression in C. fructicola via RT-qPCR, which should be useful for future functional studies of target genes in C. fructicola.

Identification and Analysis of Reference and Tissue-Specific Genes in Bitter Gourd Based on Transcriptome Data

Accurate and standardized quantification of reverse transcription PCR (qRT-PCR) results relies on the use of a dependable reference gene. The precise control of transgene expression in terms of both spatial and temporal aspects necessitates the utilization of tissue-specific gene promoters. However, the identification of stable reference genes across various tissues, particularly in fruits at different ripening stages, as well as tissue-specific genes in bitter gourds, remains largely unexplored. In this study, we employed RNA-Seq-based transcriptome datasets obtained from nine tissues to comprehensively screen for new reference genes (NRGs) and tissue-specific genes. Through the utilization of five algorithms in conjunction with qRT-PCR analysis, we successfully identified two highly stable reference genes, namely HMG1/2 and PHOS32, from a pool of 11 NRGs and five traditional reference genes (TRGs). To validate their reliability, we performed expression pattern analysis of two genes associated with fruit ripening (McACO1 and McACO2) using HMG1/2 and PHOS32, as well as an unstable reference gene, HSCP2. Furthermore, we conducted qRT-PCR validation of 12 tissue-specific genes using HMG1/2 as the reference gene. This study not only contributes to the precise normalization of target genes in bitter gourd but also provides a solid foundation for regulating transgenes through the utilization of suitable tissue-specific promoters.

Correction: Liu et al. The Genome-Wide Identification of Stable Internal Reference Genes Related to Delayed Spoilage for Accurate qRT-PCR Normalization in Ethephon-Treated Pueraria thomsonii Benth. Horticulturae 2023, 9, 1014

In the original publication [...]

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses

Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean–Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species (Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Identification of Accurate Reference Genes for qRT-PCR Analysis of Gene Expression in Eremochloa ophiuroides under Multiple Stresses of Phosphorus Deficiency and/or Aluminum Toxicity.

Centipedegrass (Eremochloa ophiuroides (Munro.) Hack.) is a species originating in China and is an excellent warm-season turfgrass. As a native species in southern China, it is naturally distributed in the phosphorus-deficient and aluminum-toxic acid soil areas. It is important to research the molecular mechanism of centipedegrass responses to phosphorus-deficiency and/or aluminum-toxicity stress. Quantitative Real-Time PCR (qRT-PCR) is a common method for gene expression analysis, and the accuracy of qRT-PCR results depends heavily on the stability of internal reference genes. However, there are still no reported stable and effective reference genes for qRT-PCR analysis of target genes under the acid-soil-related stresses in different organs of centipedegrass. For scientific rigor, the gene used as a reference for any plant species and/or any stress conditions should be first systematically screened and evaluated. This study is the first to provide a group of reliable reference genes to quantify the expression levels of functional genes of Eremochloa ophiuroides under multiple stresses of P deficiency and/or aluminum toxicity. In this study, centipedegrass seedlings of the acid-soil-resistant strain 'E041' and acid-soil-sensitive strain 'E089' were used for qRT-PCR analysis. A total of 11 candidate reference genes (ACT, TUB, GAPDH, TIP41, CACS, HNR, EP, EF1α, EIF4α, PP2A and actin) were detected by qRT-PCR technology, and the stability of candidate genes was evaluated with the combination of four internal stability analysis software programs. The candidate reference genes exhibited differential stability of expression in roots, stems and leaves under phosphorus-deficiency and/or aluminum-toxicity stress. On the whole, the results showed that GAPDH, TIP41 and HNR were the most stable in the total of samples. In addition, for different tissues under various stresses, the selected reference genes were also different. CACS and PP2A were identified as two stable reference genes in roots through all three stress treatments (phosphate deficiency, aluminum toxicity, and the multiple stress treatment of aluminum toxicity and phosphate deficiency). Moreover, CACS was also stable as a reference gene in roots under each treatment (phosphate deficiency, aluminum toxicity, or multiple stresses of aluminum toxicity and phosphate deficiency). In stems under all three stress treatments, GAPDH and EIF4α were the most stable reference genes; for leaves, PP2A and TIP41 showed the two highest rankings in all three stress treatments. Finally, qRT-PCR analysis of the expression patterns of the target gene ALMT1 was performed to verify the selected reference genes. The application of the reference genes identified as internal controls for qRT-PCR analysis will enable accurate analysis of the target gene expression levels and expression patterns in centipedegrass under acid-soil-related stresses.

Systematic identification of reference genes for qRT-PCR of Ardisia kteniophylla A. DC under different experimental conditions and for anthocyanin-related genes studies.

Ardisia kteniophylla A. DC, widely known as folk medicinal herb and ornamental plant, has been extensively investigated due to its unique leaf color, anti-cancer and other pharmacological activities. The quantitative real-time PCR (qRT-PCR) was an excellent tool for the analysis of gene expression with its high sensitivity and quantitative properties. Normalizing gene expression with stable reference genes was essential for qRT-PCR accuracy. In addition, no studies have yet been performed on the selection, verification and stability of internal reference genes suitable for A. kteniophylla, which has greatly hindered the biomolecular researches of this species. In this study, 29 candidate genes were successfully screened according to stable expression patterns of large-scale RNA seq data that from a variety of tissues and the roots of different growth stages in A. kteniophylla. The candidates were then further determined via qRT-PCR in various experimental samples, including MeJA, ABA, SA, NaCl, CuSO4, AgNO3, MnSO4, CoCl2, drought, low temperature, heat, waterlogging, wounding and oxidative stress. To assess the stability of the candidates, five commonly used strategies were employed: delta-CT, geNorm, BestKeeper, NormFinder, and the comprehensive tool RefFinder. In summary, UBC2 and UBA1 were found to be effective in accurately normalizing target gene expression in A. kteniophella regardless of experimental conditions, while PP2A-2 had the lowest stability. Additionally, to verify the reliability of the recommended reference genes under different colored leaf samples, we examined the expression patterns of six genes associated with anthocyanin synthesis and regulation. Our findings suggested that PAP1 and ANS3 may be involved in leaf color change in A. kteniphella. This study successfully identified the ideal reference gene for qRT-PCR analysis in A. kteniphella, providing a foundation for future research on gene function, particularly in the biosynthesis of anthocyanins.

Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Selection and Validation of Reference Genes for qRT-PCR Analysis of Gene Expression in Tropaeolum majus (Nasturtium)

Tropaeolum majus (nasturtium) is an important ornamental and medicinal plant due to its colorful flowers, shield-shaped leaves, and richness in mineral elements and bioactive compounds. However, the key genes related to these important biological traits, as well as their expression patterns and functions, remain obscure. In this study, to choose appropriate reference genes for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, we screened 14 candidate genes from the transcriptome of T. majus and evaluated their expression stability. Through evaluation with four commonly used algorithms (geNorm, NormFinder, BestKeeper, and RefFinder), EXP1, EXP2, and TUB6 were found to be the most stably expressed genes among different organs, while EXP1 combined with CYP2 was identified as the optimal reference gene combination for seeds at different development stages. For all the tested samples, EXP1, EXP2, CYP2, and ACT2 were the most suitable reference genes. Moreover, the target gene KCS11 involved in very-long-chain fatty acid biosynthesis was employed to confirm the most and least stable reference genes in different organs, seeds at different development stages, and all the tested samples. The expression profiles of KCS11 were similar, with minor differences based on the analysis of different stable reference genes (either alone or in combination), while the expression profiles were diverse and the relative expression level was overestimated when using the least stable ones. These results suggest that the appropriate selection of reference genes is critical for the normalization of gene expression. Furthermore, the reference genes screened in this study will greatly improve the accuracy of the qRT-PCR quantification of candidate genes involved in the many biological characteristics of nasturtium.

Screening and validation of the optimal panel of reference genes in colonic epithelium and relative cancer cell lines

Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common method to determine mRNA expression, and Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that a panel of reference genes for RT-qPCR is conducive to obtaining accurate results. This study aimed to screen and verify the optimal panel of reference genes in colorectal cancer (CRC) and normal colonic cell lines. In the study, eight candidate reference genes (GAPDH, ACTB, 18S, PPIA, B2M, SDHA, GUSB, and YWHAZ) were selected for RT-qPCR to detect their expression in NCM460, HT29, HCT116, SW480, SW620, DLD-1, LOVO and RKO cell lines. The stability of reference genes and the optimal panel were evaluated by geNorm, NormFinder, and BestKeeper software. As results, the expression levels of candidate reference genes differed in the colonic epithelial cell lines, and the number of optimal panel of reference genes is two. B2M and YWHAZ were the two most stable reference genes for NCM460, HCT116, SW620, LOVO, and RKO cell lines, while only one of B2M and YWHAZ was most stable in HT29 and SW480 cells. In DLD-1 cells, the stability of B2M and YWHAZ ranked 3rd and 6th, PPIA and GUSB were the most stable two. Furthermore, the YWHZA + B2M performed smaller intragroup differences than other panel or single reference gene. In conclusion, this study indicates the optimal panel of reference genes is YWHZA + B2M for the NCM460, HCT116, SW620, LOVO, RKO, SW480, and HT29 cell lines, but it is PPIA + GUSB in DLD-1 cell lines.

MiR-26a/30d/152 are reliable reference genes for miRNA quantification in skin wound age estimation.

The small size of miRNAs makes them less susceptible to post-mortem autolysis or putrefaction, leading to their potential use in wound age estimation.Studying miRNAs as biological indicators of skin wound age estimation requires the selection and validation of stable reference genes because commonly used reference genes, such as U6, ACTB, GAPDH, 5S, 18S, and LC-Ogdh, are not stable.miR-26a/30d/152 are stable and reliable as reference genes and their use in combination is a recommended normalization strategy for miRNA quantitative analysis in wounded skin.