Tujia ethnic group medicine Xuetong is derived from Kadsura heteroclita, the stem of which has the medicinal value for anti-rheumatoid arthritis, liver protection, anti-tumor, anti-oxidation effects, and has been widely used in Hunan and Guangdong in China. The selection of reliable and stable reference genes is the basis for subsequent molecular research on K. heteroclita. In this study, GAPDH, TUA, Actin, UBQ, EF-1α, 18S-rRNA, CYP, UBC, TUB, H2A, and RPL were selected as candidate reference genes in Kadsura heteroclita. The gene expression levels of the 11 candidate reference genes of K. heteroclita in its 6 different parts(stem-inside of the cambium, stem-outside of the cambium, fruit, flower, root, and leaf) and under different intervention conditions [drought stress, salt stress, and methyl jasmonate(MeJA) treatment] were detected by quantitative real-time polymerase chain reaction(qRT-PCR). The expression stability of the 11 candidate reference genes was comprehensively analyzed and evaluated by geNorm, NormFinder, ΔCT algorithm, and RefFinder software. The results showed that the expression of UBC and RPL was relatively stable in 6 different parts, and UBC and GAPDH genes were relatively stable under different intervention conditions. To verify the reliability of reference genes for K. heteroclita, this study further examined the relative expression levels of KhFPS, KhIDI, KhCAS, KhSQE, KhSQS, KhSQS-2, KhHMGS, KhHMGR, KhMVD, KhMVK, KhDXR, KhDXS, KhPMVK, and KhGGPS in different parts and under different intervention conditions, which might relate to the synthesis of the main component(Xuetongsu) of K. heteroclita. The results showed that with UBC and RPL or UBC and GAPDH as the reference genes, the expression trends of these 14 genes were basically consistent in different parts or under different intervention conditions for K. heteroclita. In conclusion, UBC can be used as a reference gene of K. heteroclita for its different parts and different intervention conditions, which lays a foundation for further research on the biosynthetic pathway of main components in K. heteroclita.
Real-Time Polymerase Chain Reaction/methods* ; Reference Standards ; Gene Expression Regulation, Plant ; Gene Expression Profiling ; Plant Proteins/metabolism* ; Drugs, Chinese Herbal

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.
Real-Time Polymerase Chain Reaction/methods* ; Paeonia/genetics* ; Actins/genetics* ; Reproducibility of Results ; Transcriptome ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Reference Standards ; Gene Expression Profiling/methods*

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.
Genes, Essential ; Gelsemium/genetics* ; Peptide Elongation Factor 1/genetics* ; Transcriptome ; Gene Expression Profiling/methods* ; Alkaloids ; Real-Time Polymerase Chain Reaction/methods* ; Reference Standards

OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/physiology* ; Phenotype ; RNA/isolation & purification* ; Real-Time Polymerase Chain Reaction/standards* ; Rho Guanine Nucleotide Exchange Factors/metabolism* ; Sincalide/analysis* ; Spectrophotometry/methods* ; Zebrafish/physiology*

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.
Artemisia/genetics* ; Gene Expression Profiling ; Genes, Plant/genetics* ; Plant Leaves/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Transcriptome

To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.
Fallopia multiflora ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Genes, Plant/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Reproducibility of Results

Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.
Gene Expression Profiling ; Gene Expression Regulation, Plant ; Genes, Plant/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.
Aconitum ; Gene Expression Profiling ; Genes, Plant/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction

Screening the reference genes that were stably expressed under different light intensities for Viola yedoensis could provide reference for the related molecular research. In this study, 11 candidate reference genes were detected by RT-qPCR for tissues of V. yedoensis treated with different light intensities. Ge Norm, Norm Finder, Best Keeper, and Ref Finder website were used to comprehensively evaluate the reference genes, and verify the stability of the reference gene based on CAT1. Finally, the ideal reference gene was determined. The results showed that CYP, Actin, and SAMDC had small Ct value ranges and stable expression. Ge Norm demonstrated that CYP, SAMDC, and Actin were suitable reference genes. Norm Finder showed that the expression of α-TUB was the most stable. Best Keeper recommended CYP, Actin, and SAMDC as reference genes. Ref Finder assessed that SAMDC, CYP, α-TUB, and Actin had better stability, while GAPDH had the worst stability. The expression trend of CAT1 gene was consistent when calibrated with SAMDC, CYP, and Actin, while it was quite different from that calibrated with GAPDH. In summary, SAMDC, CYP, and Actin can be used as ideal reference genes for the gene expression profiling of V. yedoensis under different light intensities.
Gene Expression Profiling ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Viola

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.
DNA Primers ; Formaldehyde ; Gene Expression Profiling ; Humans ; MicroRNAs/analysis* ; Myocardium ; Paraffin Embedding ; RNA/analysis* ; Real-Time Polymerase Chain Reaction/standards*