Startseite Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia
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Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia

  • Jinhua Liu ORCID logo , Chuang Yang , Mingzhu Bai , Feng Yan , Haiying Qin , Ruigang Wang , Yongqing Wan EMAIL logo und Guojing Li EMAIL logo
Veröffentlicht/Copyright: 14. September 2022
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Open Life Sciences
Aus der Zeitschrift Open Life Sciences Band 17 Heft 1

Abstract

Reverse transcription quantitative PCR (RT-qPCR) is a technique widely used to investigate the expression of genes. An appropriate reference gene (RG) is essential for RT-qPCR analysis to obtain accurate and reliable results. Caragana intermedia plays an important role in afforestation as a bush. However, due to the lack of appropriate RGs, the research on development-related genes is limited. In this study, the selection for suitable RGs of different organs at various development stages to normalize the results of RT-qPCR about development-related genes was performed. To test the expression stability across all samples, we used the software algorithms such as geNorm, NormFinder, BestKeeper, and RefFinder to evaluate all the candidate RGs. Our results showed that CiEF1α was the most stable RG with little fluctuation among all samples. In addition, CiGAPDH in roots, CiSKIP1 in stems and leaves, and CiEF1α in different organs were selected as the most stable RGs. To confirm the applicability of the most stable RGs, the relative expression of CiWRKY17 was normalized using different candidate RGs. Taken together, our research laid a foundation for the study of development-related genes in C. intermedia.

Keywords: Caragana intermedia ; development stage; RT-qPCR; reference gene

1 Introduction

Caragana intermedia, commonly known as a bush, belongs to the legume family and is widely distributed in north and northwest China along with semi-fixed or fixed sand dunes, barren land, and loess hills. C. intermedia has high ecological value via playing an important role in afforestation [1]. To make the bush perform its role, it is essential to study the growth and development of C. intermedia to achieve the goal of cultivating healthy seedlings quickly and transferring them efficiently for afforestation.

Today, with the wide recognition of the important role of growth and development of plants, there have been many studies about development [2,3], and the research on development-relevant genes in C. intermedia has also made some progress [4]. In order to better uncover the function of these genes, it is pivotal to analyze their spatio-temporal expression [5]. Reverse transcription quantitative PCR (RT-qPCR) was often used to study the expression of genes because of its high throughput, specificity, and sensitivity [6–8], but its accuracy is difficult to guarantee due to changes in mRNA quantity and quality, and other reasons. Using relatively stable reference genes (RGs) can be a good solution to this problem. Therefore, it is necessary to select appropriate RGs.

Under ideal conditions, the RGs should be relatively stable, but some reports indicated that the expression of these so-called RGs could fluctuate in different organs and at various stages in plants [9–12]. For example, the commonly used RGs, such as Actin (ACT) [13], Beta-tubulin (TUB) [14], Elongation factor 1-α (EF1α) [10,15], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were often chosen for normalization of RT-qPCR in plants, but EF1α showed no expression stability in the root of Prunus spp. [16], and TUB was one of the least stable genes in the cells elicited with MeJA of Cichorium intybus [17]. So it is quite necessary to screen RGs for their suitability for different experimental designs. Owing to no perfect method to evaluate the stability of RGs, four different analytical software, such as geNorm [18], NormFinder [19], BestKeeper [20], and RefFinder [23], were usually used to identify the suitable RGs.

Previous studies showed that the RGs of C. intermedia [21] had been screened under various abiotic stresses. However, these RGs were not screened in different organs or at various development stages, so this study aims to select relatively stable RGs for the reference of the development-related genes of C. intermedia. First, we selected 11 commonly used RGs, such as CiACT, CiCAP (Cyclase-associated protein), CiEF1a, CiGAPDH, CiSKIP1/SKIP5-1/SKIP5-2 (F-box proteins), CiTUA (Alpha-tubulin), CiTUB/TUB3 (Beta-tubulins), and CiUBQ (Ubiquitin), which were proved to be relatively stable in its homologous species, C. korshinskii [22], and the expression level of these RGs were analyzed among samples from different organs or at various development stages by RT-qPCR. Next, all these selected RGs were ranked using geNorm, NormFinder, BestKeeper, and RefFinder software. Finally, CiWRKY17, which had been proved to express in different organs, might be involved in the growth and development of C. intermedia [4], was selected to validate these RGs. The above results will provide the most appropriate RGs for further study of development-related genes in C. intermedia.

2 Materials and methods

2.1 Collection of plant materials

The samples were collected in the field located in Liangcheng County, Ulanchabu City, Inner Mongolia, China, with north latitude 41°23′ and an east longitude 111°41′. Totally 69 samples from seven different stages of development (Figure 1), and seven organs, including root, stem, leaf, bud, flower, young pods, and young seeds, were collected as indicated in Figure 1 and Table 1. Each sample was taken from an even mixture of the same tissues from three different plants, and three biological replicates were performed. All samples were immediately frozen in liquid nitrogen for RNA extraction in the follow-up steps.

Figure 1 Different developmental stages of the samples.
Figure 1

Different developmental stages of the samples.

Table 1

The number of samples of different organs

Stage Root Stem Leaf Bud Flower Pod Seed
The samples S1 3 3 — — — — —
S2 3 3 — — — — —
S3 3 3 3 3 — — —
S4 3 3 3 — 3 — —
S5 3 3 3 — — 3 —
S6 3 3 3 — — — 3
S7 3 3 3 — — — —

2.2 RNA isolation and cDNA synthesis

RNA extraction and reverse transcription were performed using TaKaRa MiniBEST Plant RNA Extraction Kit and TaKaRa PrimeScriptTMRT reagent Kit with gDNA Eraser, respectively. RNA was measured using an ultraviolet spectrophotometer (Model DU800), and the ratio of A260 nm/A280 nm and A260 nm/A230 nm was calculated to check the purity and the concentration (μg/mL) of the extracted RNA. RNA integrity was assessed using electrophoresis of the extracted RNA on a 1.0% (w/v) agarose gel. Any RNA sample with an A260/A280 ratio between 1.8 and 2.2 and an A260/A230 ratio greater than 2.0 was used for subsequent experiments. One microgram of total RNA from each sample was used to synthesize the first-strand cDNAs using the above-mentioned reverse transcription Kit according to the manufacturer’s instructions. The cDNA acquired by reverse transcription was then diluted to 16-fold and used as the template for RT-qPCR.

2.3 Primer and RT-qPCR

The sequences for the primers of the selected 11 RGs were obtained from our previously published article [22] and are listed in Table S1. The primers’ sequence, amplification efficiency, the regression coefficient, R 2 value, and the melting curve are also listed in Table S1 or in Figure S1. The cDNA was amplified using TB Green qPCR Master Mix (TaKaRa) with a Roche LightCycler 480 system. The thermal cycling program was 95°C for 60 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 15 s. Each RT-qPCR reaction was performed with three technical replicates. The Ct values, which took the mean value of three technical replicates, were pooled for the RG evaluation.

2.4 Data analysis

Stability analysis of the RGs was assessed using the Excel-based geNorm [18], NormFinder [19], BestKeeper [20], and RefFinder [23], which were the most widely used software to screen RG by RT-qPCR.

For geNorm and NormFinder algorithms, the raw Ct values from each sample were converted into relative quantity (RQ) values using the formula 2−ΔCt (ΔCt = each Ct value − the minimum Ct value) [24]. The geNorm program first calculated an expression stability M-value for each gene, and M-values below 1.5 were supposed to be stably expressed, and a lower M-value indicated a more stable expression [18]. Moreover, the value of “n” was the optimal number of RGs when the pairwise value of variation (V n /V n+1) was below a cutoff value of 0.15 [18]. NormFinder was used to assess the stability of RGs based on the ANOVA model [19].

For BestKeeper and RefFinder programs, the raw Ct values were directly analyzed. BestKeeper examined the ranking of RGs based on the calculation of the variance and the standard deviation (SD) for each gene. Any gene with an SD value less than 1.0 was recommended as a gene with stable expression [20]. RefFinder, a user-friendly and web-based comprehensive tool, was developed for selecting RGs, and integrated the currently available computational programs to compare the rank of the tested RGs [23].

2.5 Validation of RGs

CiWRKY17 was selected to verify the most stable RG, the most stable RG combination, and the worst RG by RT-qPCR. Each qPCR reaction was performed with three technical replicates. The experiment was repeated three times (with three biological replicates), and the results were consistent. Finally, the data were calculated using the 2−ΔΔCt method.

3 Results

3.1 Expression profiles of the RGs

The expression level of the 11 RGs was analyzed across all samples via determination of the Ct value, and lower the Ct value the higher the gene expression level was. The Ct value of these genes ranged from 18 to 34 in all studied samples, with most of them between 20 and 26, and the smaller the range of the Ct value the more stable the RG was (Figure 2).

Figure 2 Distribution of the Ct values of the 11 RGs among all samples: the box plot graph shows the maxima, minima, medians, and the 25th/75th percentile.
Figure 2

Distribution of the Ct values of the 11 RGs among all samples: the box plot graph shows the maxima, minima, medians, and the 25th/75th percentile.

To evaluate the relative stability value of RGs, we took the average Ct value and Ct value range. On the one hand, CiACT (with an average Ct of 21.15) showed the highest expression level, whereas CiSKIP5-1 (with an average Ct of 24.9), UBQ (with an average Ct of 24.9), and CiCAP (with an average Ct of 25.2) showed relatively low expression level (Figure 2). On the other hand, the six genes with the minimum Ct value range were CiSKIP1 (4.61), CiGAPDH (5.67), CiSKIP5-2 (5.83), CiEF1α (6.14), CiTUA (8.12), and CiSKIP5-1 (8.57) (Figure 2). In brief, based on both the higher expression level and the lower Ct value range, CiSKIP1, CiGAPDH, CiSKIP5-2, and CiEF1α were more appropriate as RGs.

3.2 Stability analysis by geNorm

According to geNorm analysis, the M-values of all tested RGs were below 1.5, indicating that they were relatively stable, and the lowest M-value indicated the highest stability [18]. Among all of the tested samples (Figure 3e), CiSKIP1 was found to be the stable RGs successively, while CiSKIP5-2 and CiSKIP5-1 represented the most stable RG combination. On the contrary, CiTUB3 was found to be the most inappropriate RG based on their fluctuating expression levels. In addition, these screening results of RGs varied in different organ samples (Figure 3a–d).

Figure 3 Expression stability measurement (M) and pairwise variation (V) analysis of the 11 selected RGs in samples from different organs or at various stages using geNorm: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates; and (f) pairwise variation (V) analysis.
Figure 3

Expression stability measurement (M) and pairwise variation (V) analysis of the 11 selected RGs in samples from different organs or at various stages using geNorm: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates; and (f) pairwise variation (V) analysis.

The optimal number of RGs for normalizing RT-qPCR data was determined by calculating the pairwise variations V n /V n+1 in the geNorm program, and when the value of V n /V n+1 is less than 0.15, the n-value is the optimal number of RGs [18]. As shown in Figure 3f, since the values of V2/3 and V3/4 were greater than 0.15 from left to right, and the value of V4/5 was below the cutoff value of 0.15, these four RGs were ideal for normalizing RT-qPCR data in all samples.

3.3 Stability analysis by NormFinder

The NormFinder was similar to geNorm in algorithm to calculate different M-values of RGs. The difference was that NormFinder selected only one optimal gene, while geNorm selected two or more genes. The results calculated with NormFinder (Figure 4) showed that CiEF1α was the most stable gene in all the tested samples, while CiTUB3 was considered to be a weakly stable gene, and there were some differences in different organ samples.

Figure 4 Stability value of the 11 RGs calculated by NormFinder: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates.
Figure 4

Stability value of the 11 RGs calculated by NormFinder: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates.

3.4 Stability analysis by BestKeeper

BestKeeper examined the ranking of RGs based on the calculation of the SD value for each gene. Any gene with an SD value less than 1.0 was recommended as a gene with stable expression, and the lowest SD value was the highest stability of a gene. According to the above principles, the result showed CiSKIP1 was the most stable gene, and CiACT was the least stable gene in all tested samples, and the results differed in different organ samples (Figure 5a–c).

Figure 5 Stability value of the 11 RGs calculated by BestKeeper: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates.
Figure 5

Stability value of the 11 RGs calculated by BestKeeper: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates.

3.5 Comprehensive stability analysis of RGs by RefFinder

In order to comprehensively evaluate the stability of RGs, we made a comprehensive ranking by RefFinder to get the most stable RGs. As shown in Figure 6, CiEF1α was the most stable RGs with little fluctuation; moreover, CiACT was the least stable RGs due to large expression of fluctuations in all samples. Similarly, CiGAPDH in roots, CiSKIP1 in stems and leaves, and CiEF1α in different organs were found to be the most stable RGs; CiTUA in roots and CiTUB3 in stems, leaves, and different organs were not suitable to be used as RGs.

Figure 6 The comprehensive ranking of the 11 RGs by RefFinder: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates.
Figure 6

The comprehensive ranking of the 11 RGs by RefFinder: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (including buds, flowers, pods, roots, stems, leaves, and seeds samples), buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6; (e) all samples from different developmental stages; (a–e) represent all samples containing three biological replicates.

3.6 Verification of the selected RGs

In order to validate the identified RGs and to demonstrate the application of the stable RGs selected by geNorm, NormFinder, BestKeeper, and RefFinder under the studied conditions, the transcript profile of CiWRKY17, which had been confirmed to express in leaf, root, and stem previously [4], was assayed again using the RGs (including the least and most stable RGs and their combinations). As shown in Figure 7, using the 2−ΔΔCt method, the expression level of CiWRKY17 was similar when we used the most stable RG and RG combination to normalize the RT-qPCR data. However, the results varied using the least stable RG.

Figure 7 Effect of different RGs on normalization of the relative expression of CiWRKY17: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6); (a–d) green, blue, and red represent the most stable RG, the most stable RG combination, and the worst RG, respectively; (a–d) represent samples containing three biological replicates (data were presented as means ± SE of three independent biological replicates; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by Student’s t-test).
Figure 7

Effect of different RGs on normalization of the relative expression of CiWRKY17: (a) roots; (b) stems; and (c) leaves from different developmental stages; (d) different organs (buds collected from the stage of S3, flowers collected from S4, pods collected from S5, and roots, stems, leaves, and seeds collected from S6); (a–d) green, blue, and red represent the most stable RG, the most stable RG combination, and the worst RG, respectively; (a–d) represent samples containing three biological replicates (data were presented as means ± SE of three independent biological replicates; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by Student’s t-test).

4 Discussion

The powerful technique RT-qPCR has been widely used for the detection and quantification of gene expression in plants. In order to interpret RT-qPCR data accurately and reliably, appropriate RGs are essential. Reports on several plant species, such as Undaria pinnatifida [25], Raphanus sativus L. [26], Lycoris aurea [27], Lagerstroemia indica and L. speciosa [28], Eucommia ulmoides Oliver [29], Pyrus L. [30], and Davidia involucrata Baill. [31], had shown the importance of validating appropriate RGs for normalizing RT-qPCR data. In addition, some studies have shown that the best RG was different for different samples of organs or experimental conditions [26,29,32]. RGs of C. intermedia [21] had been screened under various abiotic stresses but had not been screened in different organs collected from different developmental stages, which led to difficulties in the normalization of the development-related gene. Therefore, it is particularly important to screen RGs in different organs at various development stages.

Current studies showed no perfect analysis software to evaluate the stability of RGs. In order to obtain the stable RG, we selected four kinds of analysis software to evaluate. Among them, geNorm [18], NormFinder [19], and BestKeeper [20] obtained the best RGs, respectively: CiEF1α, CiGAPDH, and CiSKIP1 in roots; CiUBQ, CiSKIP1, and CiGAPDH in stems; CiSKIP5-2, CiEF1α, and CiCAP in leaves; CiUBQ, CiTUA, and CiSKIP1 in different organs; and CiSKIP1, CiEF1α, and CiSKIP1 in all samples. The above results indicated that different analysis software indeed screened different RGs, which might be caused by the different algorithms of this software, and this might also be the case in other reports [29,33,34]. Since different software came to a diverse conclusions, in order to obtain the best RG, we chose RefFinder [22] to integrate these conclusions and finally concluded that the best RG was CiGAPDH in roots, CiSKIP1 in stems, CiSKIP1 in leaves, CiTUB in different organs, and CiEF1α in all samples. In addition, geNorm could obtain the most suitable number of RGs according to V n /V n+1 [18]. For example, V4/V5 was less than 0.15 in all samples, indicating that four RGs were the most suitable. However, considering that multiple RGs would make the experiment more complicated and time-consuming, 1–2 RG was suitable to normalize the target genes for convenient operation. Therefore, according to the analysis results of geNorm, the optimal combination of RGs was obtained: SKIP5-1/SKIP5-2 in roots, SKIP5-1/SKIP5-2 in stems, SKIP5-1/SKIP1 in leaves, TUA/EF1α in different organs, and TUB/EF1α in all samples.

To sum up, we found that the optimal RGs varied in different organ samples, and the optimal combination of RGs was different except for root and stem. In addition, previous studies on the selection of plant RGs mainly focused on different hormones [11,35], stress treatments [29,36,37], and different tissues/organs [9,12,38], but there were no studies on the selection of plant RGs in different organs collected from various development stages. This study aimed to screen the RGs of different organs at different developmental periods. We also used the most and least stable RGs, and their combination to normalize the transcript of a known gene, CiWRKY17, to evaluate the practicality of the selected RGs, and the results indicated that the selected RGs were reliable.

5 Conclusion

In conclusion, the 11 RGs were systematically selected and evaluated using RT-qPCR in seven organ types and at seven different developmental stages, using geNorm, NormFinder, BestKeeper, and RefFinder software. Then the selected RGs were further validated by analysis of the CiWRKY17 expression in different organs. And the best RGs and the best combination of RGs were obtained. This study will improve the accuracy of the RT-qPCR results and lay the foundation for future studies on development-related genes of C. intermedia.

Acknowledgments

We deeply appreciate Martyn Rittman, Ph.D., English Editing Manager of Multidisciplinary Digital Publishing Institute, for polishing the manuscript carefully.

  1. Funding information: This work was financially supported by the High-level Talents Research Initiation Project Mission of Inner Mongolia Agricultural University (No. NDYB2018-61), National Natural Science Foundation of China (No. 31860217), the University Scientific and Technological Innovation Team Project of Inner Mongolia (NMGIRT2222) the Natural Science Foundation of Inner Mongolia Autonomous Region (2022MS03035), and Ordos Science & Technology Plant (2022EEDSKJZDZX017).

  2. Author contributions: J.L. carried out the experiment and prepared the draft of this manuscript. G.L. and Y.W. conceived and designed the experiments. R.W. and M.B. prepared the materials. C.Y., H.Q., and F.Y. contributed to the data analysis. All authors read and approved the final manuscript.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2021-12-21
Revised: 2022-04-28
Accepted: 2022-06-14
Published Online: 2022-09-14

© 2022 Jinhua Liu et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

Artikel in diesem Heft

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  2. Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits
  3. The mother of all battles: Viruses vs humans. Can humans avoid extinction in 50–100 years?
  4. Knockdown of G1P3 inhibits cell proliferation and enhances the cytotoxicity of dexamethasone in acute lymphoblastic leukemia
  5. LINC00665 regulates hepatocellular carcinoma by modulating mRNA via the m6A enzyme
  6. Association study of CLDN14 variations in patients with kidney stones
  7. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook
  8. Regulation of miR-30b in cancer development, apoptosis, and drug resistance
  9. Informatic analysis of the pulmonary microecology in non-cystic fibrosis bronchiectasis at three different stages
  10. Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway
  11. Characterization of intestinal microbiota and serum metabolites in patients with mild hepatic encephalopathy
  12. Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis
  13. Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote
  14. Biomedical evaluation of antioxidant properties of lamb meat enriched with iodine and selenium
  15. Intravenous infusion of the exosomes derived from human umbilical cord mesenchymal stem cells enhance neurological recovery after traumatic brain injury via suppressing the NF-κB pathway
  16. Effect of dietary pattern on pregnant women with gestational diabetes mellitus and its clinical significance
  17. Potential regulatory mechanism of TNF-α/TNFR1/ANXA1 in glioma cells and its role in glioma cell proliferation
  18. Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin
  19. Quercetin inhibits cytotoxicity of PC12 cells induced by amyloid-beta 25–35 via stimulating estrogen receptor α, activating ERK1/2, and inhibiting apoptosis
  20. Nutrition intervention in the management of novel coronavirus pneumonia patients
  21. circ-CFH promotes the development of HCC by regulating cell proliferation, apoptosis, migration, invasion, and glycolysis through the miR-377-3p/RNF38 axis
  22. Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma
  23. Lacunar infarction aggravates the cognitive deficit in the elderly with white matter lesion
  24. Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats
  25. Comparison of axon extension: PTFE versus PLA formed by a 3D printer
  26. Elevated IL-35 level and iTr35 subset increase the bacterial burden and lung lesions in Mycobacterium tuberculosis-infected mice
  27. A case report of CAT gene and HNF1β gene variations in a patient with early-onset diabetes
  28. Study on the mechanism of inhibiting patulin production by fengycin
  29. SOX4 promotes high-glucose-induced inflammation and angiogenesis of retinal endothelial cells by activating NF-κB signaling pathway
  30. Relationship between blood clots and COVID-19 vaccines: A literature review
  31. Analysis of genetic characteristics of 436 children with dysplasia and detailed analysis of rare karyotype
  32. Bioinformatics network analyses of growth differentiation factor 11
  33. NR4A1 inhibits the epithelial–mesenchymal transition of hepatic stellate cells: Involvement of TGF-β–Smad2/3/4–ZEB signaling
  34. Expression of Zeb1 in the differentiation of mouse embryonic stem cell
  35. Study on the genetic damage caused by cadmium sulfide quantum dots in human lymphocytes
  36. Association between single-nucleotide polymorphisms of NKX2.5 and congenital heart disease in Chinese population: A meta-analysis
  37. Assessment of the anesthetic effect of modified pentothal sodium solution on Sprague-Dawley rats
  38. Genetic susceptibility to high myopia in Han Chinese population
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  40. Silencing circular RNA-friend leukemia virus integration 1 restrained malignancy of CC cells and oxaliplatin resistance by disturbing dyskeratosis congenita 1
  41. Endostar plus pembrolizumab combined with a platinum-based dual chemotherapy regime for advanced pulmonary large-cell neuroendocrine carcinoma as a first-line treatment: A case report
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  46. Effect of cell receptors in the pathogenesis of osteoarthritis: Current insights
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  48. Study of hsa_circRNA_000121 and hsa_circRNA_004183 in papillary thyroid microcarcinoma
  49. BuyangHuanwu Decoction attenuates cerebral vasospasm caused by subarachnoid hemorrhage in rats via PI3K/AKT/eNOS axis
  50. Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart
  51. Monosodium iodoacetate-induced subchondral bone microstructure and inflammatory changes in an animal model of osteoarthritis
  52. A rare presentation of type II Abernethy malformation and nephrotic syndrome: Case report and review
  53. Rapid death due to pulmonary epithelioid haemangioendothelioma in several weeks: A case report
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  144. Effects of heavy metals on bacterial community surrounding Bijiashan mining area located in northwest China
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  160. Agronomic and economic performance of mung bean (Vigna radiata L.) varieties in response to rates of blended NPS fertilizer in Kindo Koysha district, Southern Ethiopia
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  171. Erratum
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  173. Erratum to “A two-microRNA signature predicts the progression of male thyroid cancer”
  174. Retraction
  175. Retraction of “Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis”
https://doi.org/10.1515/biol-2022-0463
Schlagwörter für diesen Artikel
Caragana intermedia; development stage; RT-qPCR; reference gene
Creative Commons
BY 4.0

Artikel in diesem Heft

  1. Biomedical Sciences
  2. Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits
  3. The mother of all battles: Viruses vs humans. Can humans avoid extinction in 50–100 years?
  4. Knockdown of G1P3 inhibits cell proliferation and enhances the cytotoxicity of dexamethasone in acute lymphoblastic leukemia
  5. LINC00665 regulates hepatocellular carcinoma by modulating mRNA via the m6A enzyme
  6. Association study of CLDN14 variations in patients with kidney stones
  7. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook
  8. Regulation of miR-30b in cancer development, apoptosis, and drug resistance
  9. Informatic analysis of the pulmonary microecology in non-cystic fibrosis bronchiectasis at three different stages
  10. Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway
  11. Characterization of intestinal microbiota and serum metabolites in patients with mild hepatic encephalopathy
  12. Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis
  13. Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote
  14. Biomedical evaluation of antioxidant properties of lamb meat enriched with iodine and selenium
  15. Intravenous infusion of the exosomes derived from human umbilical cord mesenchymal stem cells enhance neurological recovery after traumatic brain injury via suppressing the NF-κB pathway
  16. Effect of dietary pattern on pregnant women with gestational diabetes mellitus and its clinical significance
  17. Potential regulatory mechanism of TNF-α/TNFR1/ANXA1 in glioma cells and its role in glioma cell proliferation
  18. Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin
  19. Quercetin inhibits cytotoxicity of PC12 cells induced by amyloid-beta 25–35 via stimulating estrogen receptor α, activating ERK1/2, and inhibiting apoptosis
  20. Nutrition intervention in the management of novel coronavirus pneumonia patients
  21. circ-CFH promotes the development of HCC by regulating cell proliferation, apoptosis, migration, invasion, and glycolysis through the miR-377-3p/RNF38 axis
  22. Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma
  23. Lacunar infarction aggravates the cognitive deficit in the elderly with white matter lesion
  24. Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats
  25. Comparison of axon extension: PTFE versus PLA formed by a 3D printer
  26. Elevated IL-35 level and iTr35 subset increase the bacterial burden and lung lesions in Mycobacterium tuberculosis-infected mice
  27. A case report of CAT gene and HNF1β gene variations in a patient with early-onset diabetes
  28. Study on the mechanism of inhibiting patulin production by fengycin
  29. SOX4 promotes high-glucose-induced inflammation and angiogenesis of retinal endothelial cells by activating NF-κB signaling pathway
  30. Relationship between blood clots and COVID-19 vaccines: A literature review
  31. Analysis of genetic characteristics of 436 children with dysplasia and detailed analysis of rare karyotype
  32. Bioinformatics network analyses of growth differentiation factor 11
  33. NR4A1 inhibits the epithelial–mesenchymal transition of hepatic stellate cells: Involvement of TGF-β–Smad2/3/4–ZEB signaling
  34. Expression of Zeb1 in the differentiation of mouse embryonic stem cell
  35. Study on the genetic damage caused by cadmium sulfide quantum dots in human lymphocytes
  36. Association between single-nucleotide polymorphisms of NKX2.5 and congenital heart disease in Chinese population: A meta-analysis
  37. Assessment of the anesthetic effect of modified pentothal sodium solution on Sprague-Dawley rats
  38. Genetic susceptibility to high myopia in Han Chinese population
  39. Potential biomarkers and molecular mechanisms in preeclampsia progression
  40. Silencing circular RNA-friend leukemia virus integration 1 restrained malignancy of CC cells and oxaliplatin resistance by disturbing dyskeratosis congenita 1
  41. Endostar plus pembrolizumab combined with a platinum-based dual chemotherapy regime for advanced pulmonary large-cell neuroendocrine carcinoma as a first-line treatment: A case report
  42. The significance of PAK4 in signaling and clinicopathology: A review
  43. Sorafenib inhibits ovarian cancer cell proliferation and mobility and induces radiosensitivity by targeting the tumor cell epithelial–mesenchymal transition
  44. Characterization of rabbit polyclonal antibody against camel recombinant nanobodies
  45. Active legumain promotes invasion and migration of neuroblastoma by regulating epithelial-mesenchymal transition
  46. Effect of cell receptors in the pathogenesis of osteoarthritis: Current insights
  47. MT-12 inhibits the proliferation of bladder cells in vitro and in vivo by enhancing autophagy through mitochondrial dysfunction
  48. Study of hsa_circRNA_000121 and hsa_circRNA_004183 in papillary thyroid microcarcinoma
  49. BuyangHuanwu Decoction attenuates cerebral vasospasm caused by subarachnoid hemorrhage in rats via PI3K/AKT/eNOS axis
  50. Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart
  51. Monosodium iodoacetate-induced subchondral bone microstructure and inflammatory changes in an animal model of osteoarthritis
  52. A rare presentation of type II Abernethy malformation and nephrotic syndrome: Case report and review
  53. Rapid death due to pulmonary epithelioid haemangioendothelioma in several weeks: A case report
  54. Hepatoprotective role of peroxisome proliferator-activated receptor-α in non-cancerous hepatic tissues following transcatheter arterial embolization
  55. Correlation between peripheral blood lymphocyte subpopulations and primary systemic lupus erythematosus
  56. A novel SLC8A1-ALK fusion in lung adenocarcinoma confers sensitivity to alectinib: A case report
  57. β-Hydroxybutyrate upregulates FGF21 expression through inhibition of histone deacetylases in hepatocytes
  58. Identification of metabolic genes for the prediction of prognosis and tumor microenvironment infiltration in early-stage non-small cell lung cancer
  59. BTBD10 inhibits glioma tumorigenesis by downregulating cyclin D1 and p-Akt
  60. Mucormycosis co-infection in COVID-19 patients: An update
  61. Metagenomic next-generation sequencing in diagnosing Pneumocystis jirovecii pneumonia: A case report
  62. Long non-coding RNA HOXB-AS1 is a prognostic marker and promotes hepatocellular carcinoma cells’ proliferation and invasion
  63. Preparation and evaluation of LA-PEG-SPION, a targeted MRI contrast agent for liver cancer
  64. Proteomic analysis of the liver regulating lipid metabolism in Chaohu ducks using two-dimensional electrophoresis
  65. Nasopharyngeal tuberculosis: A case report
  66. Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice
  67. Aberrant pulmonary immune response of obese mice to periodontal infection
  68. Bacteriospermia – A formidable player in male subfertility
  69. In silico and in vivo analysis of TIPE1 expression in diffuse large B cell lymphoma
  70. Effects of KCa channels on biological behavior of trophoblasts
  71. Interleukin-17A influences the vulnerability rather than the size of established atherosclerotic plaques in apolipoprotein E-deficient mice
  72. Multiple organ failure and death caused by Staphylococcus aureus hip infection: A case report
  73. Prognostic signature related to the immune environment of oral squamous cell carcinoma
  74. Primary and metastatic squamous cell carcinoma of the thyroid gland: Two case reports
  75. Neuroprotective effects of crocin and crocin-loaded niosomes against the paraquat-induced oxidative brain damage in rats
  76. Role of MMP-2 and CD147 in kidney fibrosis
  77. Geometric basis of action potential of skeletal muscle cells and neurons
  78. Babesia microti-induced fulminant sepsis in an immunocompromised host: A case report and the case-specific literature review
  79. Role of cerebellar cortex in associative learning and memory in guinea pigs
  80. Application of metagenomic next-generation sequencing technique for diagnosing a specific case of necrotizing meningoencephalitis caused by human herpesvirus 2
  81. Case report: Quadruple primary malignant neoplasms including esophageal, ureteral, and lung in an elderly male
  82. Long non-coding RNA NEAT1 promotes angiogenesis in hepatoma carcinoma via the miR-125a-5p/VEGF pathway
  83. Osteogenic differentiation of periodontal membrane stem cells in inflammatory environments
  84. Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway
  85. Continuous renal replacement therapy combined with double filtration plasmapheresis in the treatment of severe lupus complicated by serious bacterial infections in children: A case report
  86. Simultaneous triple primary malignancies, including bladder cancer, lymphoma, and lung cancer, in an elderly male: A case report
  87. Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine
  88. One case of iodine-125 therapy – A new minimally invasive treatment of intrahepatic cholangiocarcinoma
  89. S1P promotes corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model
  90. Early cancer detection by a targeted methylation assay of circulating tumor DNA in plasma
  91. Calcifying nanoparticles initiate the calcification process of mesenchymal stem cells in vitro through the activation of the TGF-β1/Smad signaling pathway and promote the decay of echinococcosis
  92. Evaluation of prognostic markers in patients infected with SARS-CoV-2
  93. N6-Methyladenosine-related alternative splicing events play a role in bladder cancer
  94. Characterization of the structural, oxidative, and immunological features of testis tissue from Zucker diabetic fatty rats
  95. Effects of glucose and osmotic pressure on the proliferation and cell cycle of human chorionic trophoblast cells
  96. Investigation of genotype diversity of 7,804 norovirus sequences in humans and animals of China
  97. Characteristics and karyotype analysis of a patient with turner syndrome complicated with multiple-site tumors: A case report
  98. Aggravated renal fibrosis is positively associated with the activation of HMGB1-TLR2/4 signaling in STZ-induced diabetic mice
  99. Distribution characteristics of SARS-CoV-2 IgM/IgG in false-positive results detected by chemiluminescent immunoassay
  100. SRPX2 attenuated oxygen–glucose deprivation and reperfusion-induced injury in cardiomyocytes via alleviating endoplasmic reticulum stress-induced apoptosis through targeting PI3K/Akt/mTOR axis
  101. Aquaporin-8 overexpression is involved in vascular structure and function changes in placentas of gestational diabetes mellitus patients
  102. Relationship between CRP gene polymorphisms and ischemic stroke risk: A systematic review and meta-analysis
  103. Effects of growth hormone on lipid metabolism and sexual development in pubertal obese male rats
  104. Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep
  105. Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial–mesenchymal transition in clear-cell renal cell carcinoma
  106. PHF8 promotes osteogenic differentiation of BMSCs in old rat with osteoporosis by regulating Wnt/β-catenin pathway
  107. A review of the current state of the computer-aided diagnosis (CAD) systems for breast cancer diagnosis
  108. Bilateral dacryoadenitis in adult-onset Still’s disease: A case report
  109. A novel association between Bmi-1 protein expression and the SUVmax obtained by 18F-FDG PET/CT in patients with gastric adenocarcinoma
  110. The role of erythrocytes and erythroid progenitor cells in tumors
  111. Relationship between platelet activation markers and spontaneous abortion: A meta-analysis
  112. Abnormal methylation caused by folic acid deficiency in neural tube defects
  113. Silencing TLR4 using an ultrasound-targeted microbubble destruction-based shRNA system reduces ischemia-induced seizures in hyperglycemic rats
  114. Plant Sciences
  115. Seasonal succession of bacterial communities in cultured Caulerpa lentillifera detected by high-throughput sequencing
  116. Cloning and prokaryotic expression of WRKY48 from Caragana intermedia
  117. Novel Brassica hybrids with different resistance to Leptosphaeria maculans reveal unbalanced rDNA signal patterns
  118. Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.)
  119. Phytoremediation of pollutants from wastewater: A concise review
  120. Genome-wide identification and characterization of NBS-encoding genes in the sweet potato wild ancestor Ipomoea trifida (H.B.K.)
  121. Alleviative effects of magnetic Fe3O4 nanoparticles on the physiological toxicity of 3-nitrophenol to rice (Oryza sativa L.) seedlings
  122. Selection and functional identification of Dof genes expressed in response to nitrogen in Populus simonii × Populus nigra
  123. Study on pecan seed germination influenced by seed endocarp
  124. Identification of active compounds in Ophiopogonis Radix from different geographical origins by UPLC-Q/TOF-MS combined with GC-MS approaches
  125. The entire chloroplast genome sequence of Asparagus cochinchinensis and genetic comparison to Asparagus species
  126. Genome-wide identification of MAPK family genes and their response to abiotic stresses in tea plant (Camellia sinensis)
  127. Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia
  128. Cloning and expression analysis of SERK1 gene in Diospyros lotus
  129. Integrated metabolomic and transcriptomic profiling revealed coping mechanisms of the edible and medicinal homologous plant Plantago asiatica L. cadmium resistance
  130. A missense variant in NCF1 is associated with susceptibility to unexplained recurrent spontaneous abortion
  131. Assessment of drought tolerance indices in faba bean genotypes under different irrigation regimes
  132. The entire chloroplast genome sequence of Asparagus setaceus (Kunth) Jessop: Genome structure, gene composition, and phylogenetic analysis in Asparagaceae
  133. Food Science
  134. Dietary food additive monosodium glutamate with or without high-lipid diet induces spleen anomaly: A mechanistic approach on rat model
  135. Binge eating disorder during COVID-19
  136. Potential of honey against the onset of autoimmune diabetes and its associated nephropathy, pancreatitis, and retinopathy in type 1 diabetic animal model
  137. FTO gene expression in diet-induced obesity is downregulated by Solanum fruit supplementation
  138. Physical activity enhances fecal lactobacilli in rats chronically drinking sweetened cola beverage
  139. Supercritical CO2 extraction, chemical composition, and antioxidant effects of Coreopsis tinctoria Nutt. oleoresin
  140. Functional constituents of plant-based foods boost immunity against acute and chronic disorders
  141. Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast
  142. Microbial diversity of milk ghee in southern Gansu and its effect on the formation of ghee flavor compounds
  143. Ecology and Environmental Sciences
  144. Effects of heavy metals on bacterial community surrounding Bijiashan mining area located in northwest China
  145. Microorganism community composition analysis coupling with 15N tracer experiments reveals the nitrification rate and N2O emissions in low pH soils in Southern China
  146. Genetic diversity and population structure of Cinnamomum balansae Lecomte inferred by microsatellites
  147. Preliminary screening of microplastic contamination in different marine fish species of Taif market, Saudi Arabia
  148. Plant volatile organic compounds attractive to Lygus pratensis
  149. Effects of organic materials on soil bacterial community structure in long-term continuous cropping of tomato in greenhouse
  150. Effects of soil treated fungicide fluopimomide on tomato (Solanum lycopersicum L.) disease control and plant growth
  151. Prevalence of Yersinia pestis among rodents captured in a semi-arid tropical ecosystem of south-western Zimbabwe
  152. Effects of irrigation and nitrogen fertilization on mitigating salt-induced Na+ toxicity and sustaining sea rice growth
  153. Bioengineering and Biotechnology
  154. Poly-l-lysine-caused cell adhesion induces pyroptosis in THP-1 monocytes
  155. Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection
  156. Development and validation of a predictive model for immune-related genes in patients with tongue squamous cell carcinoma
  157. Agriculture
  158. Effects of chemical-based fertilizer replacement with biochar-based fertilizer on albic soil nutrient content and maize yield
  159. Genome-wide identification and expression analysis of CPP-like gene family in Triticum aestivum L. under different hormone and stress conditions
  160. Agronomic and economic performance of mung bean (Vigna radiata L.) varieties in response to rates of blended NPS fertilizer in Kindo Koysha district, Southern Ethiopia
  161. Influence of furrow irrigation regime on the yield and water consumption indicators of winter wheat based on a multi-level fuzzy comprehensive evaluation
  162. Discovery of exercise-related genes and pathway analysis based on comparative genomes of Mongolian originated Abaga and Wushen horse
  163. Lessons from integrated seasonal forecast-crop modelling in Africa: A systematic review
  164. Evolution trend of soil fertility in tobacco-planting area of Chenzhou, Hunan Province, China
  165. Animal Sciences
  166. Morphological and molecular characterization of Tatera indica Hardwicke 1807 (Rodentia: Muridae) from Pothwar, Pakistan
  167. Research on meat quality of Qianhua Mutton Merino sheep and Small-tail Han sheep
  168. SI: A Scientific Memoir
  169. Suggestions on leading an academic research laboratory group
  170. My scientific genealogy and the Toronto ACDC Laboratory, 1988–2022
  171. Erratum
  172. Erratum to “Changes of immune cells in patients with hepatocellular carcinoma treated by radiofrequency ablation and hepatectomy, a pilot study”
  173. Erratum to “A two-microRNA signature predicts the progression of male thyroid cancer”
  174. Retraction
  175. Retraction of “Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis”
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Heruntergeladen am 21.9.2025 von https://www.degruyterbrill.com/document/doi/10.1515/biol-2022-0463/html
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