Robust identification of regulatory variants(eQTLs)using a differential expression framework developed for RNA‑sequencing

Background A gap currently exists between genetic variants and the underlying cell and tissue biology of a trait,and expression quantitative trait loci(eQTL)studies provide important information to help close that gap.However,two concerns that arise with eQTL analyses using RNA-sequencing data are normalization of data across samples and the data not following a normal distribution.Multiple pipelines have been suggested to address this.For instance,the most recent analysis of the human and farm Genotype-Tissue Expression(GTEx)project proposes using trimmed means of M-values(TMM)to normalize the data followed by an inverse normal transformation.Results In this study,we reasoned that eQTL analysis could be carried out using the same framework used for dif-ferential gene expression(DGE),which uses a negative binomial model,a statistical test feasible for count data.Using the GTEx framework,we identified 35 significant eQTLs(P<5×10^(–8))following the ANOVA model and 39 significant eQTLs(P<5×10^(–8))following the additive model.Using a differential gene expression framework,we identified 930 and six significant eQTLs(P<5×10^(–8))following an analytical framework equivalent to the ANOVA and additive model,respectively.When we compared the two approaches,there was no overlap of significant eQTLs between the two frameworks.Because we defined specific contrasts,we identified trans eQTLs that more closely resembled what we expect from genetic variants showing complete dominance between alleles.Yet,these were not identified by the GTEx framework.Conclusions Our results show that transforming RNA-sequencing data to fit a normal distribution prior to eQTL analysis is not required when the DGE framework is employed.Our proposed approach detected biologically relevant variants that otherwise would not have been identified due to data transformation to fit a normal distribution.

Differential expression and significance of 5-hydroxymethylcytosine modification in hepatitis B virus carriers and patients with liver cirrhosis and liver cancer

BACKGROUND The relationship between hepatitis B surface antigen(HBsAg)-positive carrier status and liver cancer has been extensively studied.However,the epigenetic changes that occur during progression from HBsAg-positive carrier status or cirrhosis to liver cancer are unknown.The epigenetic modification of DNA hydroxymethylation is critical in tumor development.Further,5-hydroxymethylcytosine(5hmC)is an important base for DNA demethylation and epigenetic regulation.It is also involved in the assembly of chromosomes and the regulation of gene expression.However,the mechanism of action of 5hmC in HBsAgpositive carriers or patients with cirrhosis who develop liver cancer has not been fully elucidated.AIM To investigate the possible epigenetic mechanism of HBsAg-positive carriers and hepatocellular carcinoma(HCC)progression from cirrhosis.METHODS Forty HBsAg-positive carriers,forty patients with liver cirrhosis,and forty patients with liver cancer admitted to the First People's Hospital of Yongkang between March 2020 and November 2021 were selected as participants.Free DNA was extracted using a cf-DNA kit.cfDNA was extracted by 5hmC DNA sequencing for principal component analysis,the expression profiles of the three groups of samples were detected,and the differentially expressed genes(DEGs)modified by hydroxymethylation were screened.Bioinformatic analysis was used to enrich DEGs,such as in biological pathways.RESULTS A total of 16455 hydroxymethylated genes were identified.Sequencing results showed that 32 genes had significant 5hmC modification differences between HBsAg carriers and liver cancer patients,of which 30 were upregulated and 2 downregulated in patients with HCC compared with HBsAg-positive carriers.Significant 5hmC modification differences between liver cirrhosis and liver cancer patients were identified in 20 genes,of which 17 were upregulated and 3 were downregulated in patients with HCC compared with those with cirrhosis.These genes may have potential loci that are undiscovered or unelucidated,which contribute to the development and progression of liver cancer.Analysis of gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes showed that the major signaling pathways involved in the differential genes were biliary secretion and insulin secretion.The analysis of protein interactions showed that the important genes in the protein-protein interaction network were phosphoenolpyruvate carboxykinase and solute carrier family 2.CONCLUSION The occurrence and development of liver cancer involves multiple genes and pathways,which may be potential targets for preventing hepatitis B carriers from developing liver cancer.

Cloning and Differential Expression of a 1-Aminocyclopropane-1-Carboxylate Synthase cDNA from Peach

The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach Prunus persica (L.) Batsch) fruit ripening, we cloned a full_length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1 848 bp, containing 177 bp of 5′untranslated sequence, 1 449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483_amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA_based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNApacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that ofpacs12. Pacs mRNA expressed in both green mature and ripening fruit, whilepacs12mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.

Cloning,Identification and Differential Expression Analysis of Full-length cDNA of Carp Interleukin-1β

[Objective] The research aimed to carry out the cloning,identification and differential expression analysis of carp interleukin-1β （IL-1β） cDNA. [Method] By using DD-RTPCR method,the differential expression cDNA fragments were gained. The cDNA library of carp peripheral blood leucocytes which was stimulated by the mitogen was screened,and the full length cDNA of carp IL-1β was cloned. Moreover,the sequence analysis and differential expression analysis were carried out. [Result] The positive clone which had a whole ORF that encoded 276 amino acids was obtained. The cluster analysis showed that the amino acid sequence of carp IL-1β and Japanese carp closely gathered as a branch,and the homoeology of amino acid sequence reached 95%. The clustering order was the carassius,zebra fish,pig,cattle,horse,human and mouse in turn. The differential expression analysis showed that the expression of IL-1β in the leucocytes significantly increased in the prior period （4 h） after the mitogen stimulated. But as the time went by （12 and 24 h）,it didn＇t increase in the same period. The total trend of expression amount presented the peak type. [Conclusion] The research laid the foundation for further studying the expression manner,function characteristic,regulation mechanism of IL-1β in vivo and its action mechanisms in the inflammatory reaction,emergency reaction and immune response.

Differential Expression of Immune Genes between Body Side Skin and Groin Skin of Aohan Fine Wool Sheep

[Objective] To get major genes for wool traits regulation from immune genes. [Methods] Microarray technology was used to detect differentially expressed immune genes between body side skin （more wool growing） and groin skin （no wool growing） of Aohan fine wool sheep. [Results] 46 immune genes （fold change 〉2.0） were identified and classified, and then 6 of which were selected for QPCR confir- mation. The degree of consistency of the QPCR and microarray results was 66.67%, [Conclusion] Immune privilege may participate in wool growth regulation.

Elementary study of differential expression of total soluble proteins in different life phases of Porphyra yezoensis

Total soluble proteins of different life stages, filamentous sporophytes cultivated in high temperatures, and blade gametophytes harvested in different seasons, were identified by SDS-PAGE. The types and amounts of expressed proteins also varied amongst the samples. The fewest soluble proteins were present in filamentous sporophytes. There were more types and amounts of soluble protein in conchospores than in filamentous sporophytes, but fewer than in bulgy sporophytes. More types of protein were detected in filamentous sporophytes cultivated in high temperatures than in those growing in normal situations. The most types and amounts of protein were found in blade gametophytes in all samples. Blade gametophytes harvested last year and stored at -20 ℃ showed only minor differences in expression of proteins when compared with those harvested in different seasons.

Differential Expression of MicroRNAs in Response to Drought Stress in Maize

Drought is one of the major abiotic stresses that limit maize productivity. Apart from the principal transcriptional regulation, post-transcriptional regulation mediated by microRNAs appears to be the prevalent response of plants to abiotic stress. In this study, the differential expression of microRNAs in the previously evaluated drought-tolerant inbred lines R09 under drought stress was detected by microarray hybridization. The target genes of the differentially-expressed microRNAs were predicted by bioinformatics software WMD3 for plant target gene prediction. The possible regulation of the differentially-expressed microRNAs as well as their target genes in maize response to drought stress was analysed according to Gene Ontology. Sixty-eight microRNAs in 29 microRNA families were detected to be differentially expressed in the seedling of the drought-tolerant inbred line R09, accounting for 5.97% of the total number of the probes. The expression profiles were different between the two time points of the drought stress. The functions of the genes targeted by the differentially-expressed microRNAs involve multiple physiological and biochemical pathways of response to abiotic stress, such as transcription regulation, metabolism, signal transduction, hormone stimulation, and transmembrane transport. Under drought stress, the differential expression of microRNAs regulates the expression of their target genes, resulting in multiple responses of physiological and biochemical pathways relative to drought tolerance of maize, miR156, miR159 and miR319 families may play more important roles. The different members of the same family may play similar regulation effects in most cases.

Differential Expression Analysis of Genie Male Sterility A/B Lines in Chinese Cabbage-Pak-Choi (Brassica campestris ssp. chinensis Makino)

To determine differential expression of genie male sterility A/B lines in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis Makino var. communis Tsen et Lee), we used the RNA fingerprinting technique, cDNA-AFLP analysis, in different developmental stages and different tissues. While no obvious differential expressions were observed in rosette leaves, florescence leaves, and scapes, some differential expressions were found in alabstrums of A/B lines and among leaves, scapes and alabstrums. We analyzed the al-abstrums collected in different developmental stages with 10 primer combinations. We got a unique band between middle size alabstrums and large alabstrums in B line in one of the ten pair primers, and in another one pair, one band reflecting a higher gene-expression level in A line than that in B line was obtained. No unique bands were found with the other primer combinations. The bands reflecting different gene-expression level were confirmed by Northern hybridization. The results indicated that cDNA-AFLP was a suitable tool for studying differential expression of genie male sterility in plants. SDS-polyacrylamide gel electrophoresis patterns of soluble proteins further verified the difference in A/B lines.

Differential Expression of miRNAs in Rice under High Temperature Stress

The growth and development of rice are closely related with temperature. In order to clarify the mechanism of high temperature resistance in riee, in this study, using high temperature-resistant Indian rice cultivar N22 as the experimental material, Osa-rniR159c, Osa-miR159d, Osa-miR159f, Osa-miR164d, Osa- rrdR529b and Osa-miR166h-3p obtained by high-throughput sequencing as target genes, the expression patterns of these genes in young panicles of rice under high temperature stress were analyzed by RNA-tailing and primer-extension RT-PCR, which provided theoretical basis for breeding high temperature-resistant rice eultivars.

A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z

This study was to discuss the relationship among the change in the activity of Bombyx mori carboxylesterase （BmCarE） in the midguts, the differential expression of BmCarE gene （bmcare） in the midguts, and the ability of Bombyx mori resistant to densonucleosis virus （BmDNV）, and to elucidate the molecular mechanism of resistance to BmDNV-Z. With two silkworm strains, HUABA, which is susceptible to BmDNV-Z, and BC8 （a near isogenic line of HUABA）, which is completely resistant to the same virus, as materials, the activity of BmCarE in the midgut was determined by Bio-Tek Synergy, and the differential expression of bmcare between the two strains was investigated by real-time fluorescence quantitative PCR, both at 12, 36, and 72 h post oral inoculation of the two strains with virus （hereafter referred as inoculation）. While the activity of BmCarE in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculated group, and the HUABA control group by 3.28, 2.26, and 3.02 times, respectively, with the difference being highly significant （P 〈 0.01）, there was no statistical difference among the other groups. The relative expression level of bmcare in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculation group, and the HUABA control group by 17.714, 21.76, and 15.09 times, respectively, with the difference being highly significant （P 〈 0.01）, and there was no statistical difference among other groups. The elevation of BmCarE activity and expression level of bmcare in the resistant strain at 12 h post inoculation may relate to the resistant gene （nsd/nsd） and the stimulation of BmDNV-Z. The molecular basis for the elevation of BmCarE activity in the resistant strain BC8 may be the change in the expression level of bmcare.

The impact of allelochemicals on the differential expression of symbiotic bacteria in cotton aphids

Insects have developed a good adaptive mechanism in response to environmental stresses in the long-term evolution. They have developed a helpful metabolism system to resist plant allelochemicals. Insects also harbor different kinds of symbiotic bacteria, which provide them a competitive advantage. Here, using cotton aphid as an example, we investigated the effects of four plant allelochemicals on the differential expression of symbiotic bacteria based on transcriptome data. We also studied the composition of symbiotic bacteria and function on pathway level in three kinds of aphids. We found that the bacteria have a significant role in resisting the plant allelochemicals stress and host plant selection by aphids. These results should be useful to investigate the environmental adaption mechanism of aphids in the view of symbiotic bacteria. These results would offer a new insight for improving strategy of aphids and developing new pest control systems.

Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

ESTs fragments which represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fet al kidney cDNA library. Microarray was prepared by using these novel EST fragmen ts by automatic spotting. Expression patters of 79 ESTs of novel genes from huma n fetal kidney were analyzed in fetal brain and fetal heart tissues of 20\|week\ | and 26\|week\|age fetus by performing of cDNA chip hybridization. This provide s clues for studying exact functions of the novel genes. 8 genes were obtained w hich were expressed differentially in the fetal brain and heart of 20\|week\| an d 26\|week\|age respectively. Then differentially expressed genes were identifie d by Northern analysis. The more exact function of the novel genes is under stud y.

Study on PRRSV Receptor Genes Differential Expression in Lung Tissues in Different Breeds of Pigs after Infecting with HP-PRRSV

[ Objective] In order to study the susceptibility molecular mechanism of highly pathogenic porcine reproductive and respiratory syndrome virus （ HP- PRRSV） JXA1 isolate on Tibetan pig, Zangmei pig and Yorkshire pig. [ Method ] In the study, real-time quantitative RT-PCR method was established to compare and analyze the differential expression of five porcine reproductive and respiratory syndrome virus （PRRSV） receptor genes （HSPG2, SIGLEC1, CD163, VIM and NMMHC-H A） in lung tissues in Tibetan pig, Zangmei pig and Yorkshire pig before the challenge and at the 4th ,7th and 14th days after the challenge with JXAI isolate. [ Results ] HSPG2 expression in Tibetan pig lung tissues increased significantly at the 4th and 14th days after the challenge with JXAI （ P 〈 0.05 ）, while decreased significantly at the 7th day after the challenge （P 〈 0.05 ）, HSPG2 expression in Zangmei pig lung tissues increased significantly at the 14th day after the challenge （P〈0.05）. SIGLECl expression in Tibetan pig lung tissues increased significantly at the 4th and 14th days after the infection（P 〈 0.05 ）, while SIGLEC 1 expression in Yorkshire pig decreased significantly at the 4th, 7th and 14th days after the challenge （P 〈0. 05 ）. CD163 expression in lung tissues of Tibetan pig and Zangmei pig both increased significantly at the 14th day after the challenge （P 〈 0.05 ）, while CD163 expression in lung tissues of Yorkshire pig decreased significantly at the 7th and 14th days after the challenge （ P 〈 0. 05 ）. VIM expression in lung tissues of Tibetan pig increased significantly at the 7th day after the challenge （ P 〈 0. 05 ）, while which of Yorkshire pig at the 7th day after the challenge decreased significantly （ P 〈 0. 05 ）. NMMHC-II A expression in lung tissues of Zangmei pig increased significantly at the 4th day after the challenge （ P 〈 0. 05 ）, and which of Yorkshire pig increased significantly at the 4th and 14th days after the challenge （P 〈 0. 05 ）. [ Conclusion] SIGLEC1 and VIM genes might be the potential key genes affecting the susceptibility of JXA1 isolate on Tibetan pig, Zangrnei pig and Yorkshire pig. Key words JXA1 isolate; Tibetan pig; Zangmei pig; Yorkshire pig; Porcine reproductive and respiratory syndrome virus receptor genes; Differential expression

Differential expression of microRNA clusters in bladder transitional cell carcinoma

Objective： The aim of the study was to investigate the differential expression of microRNAs （miRNAs） in bladder transitional cell carcinoma （BTC）. Methods： Fresh tissues were obtained from patients with BTC （9 cases; 3 cases with grade Ⅰ, 3 cases with grade Ⅱ, 3 cases with grade III） and those with normal bladder mucosa （3 cases） and stored in liquid nitrogen. Total RNA was extracted using TRizol reagent and RNA was quantified and quality control was performed, miRNA probes were labeled with Hy3TM fluorescence, then hybridized with a miRCUR^M array labeling kit. miRNAarrays were scanned and analyzed and the scanned result was validated using reverse transcription-polymerase chain reaction （RT-PCR）. Results： In four groups of differentially expressed genes obtained from grade Ⅰ, grade Ⅱ, grade Ⅲ, and grade Ⅰ ＋ grade Ⅱ ＋ grade Ⅲ BTC tissues compared with normal bladder mucosa, hsa-miR-29b-1＊ was upregulated, and hsa-miR-923 and hsa-miR-300 were downregulated. The hsa-miR-29b-1＊, hsa-miR-300, and hsa-miR-923 findings were confirmed by real-time RT-PCR. Conclusion： Genes that were differentially expressed between BTC and normal bladder mucosa may be involved in the pathogenesis and development of BTC, and may be useful for further studies of BTC-related genes.

Differential Expression of Wnts, β-catenin and E-cadherin in hEFs and Normal, Abnormal Karyotype hES Cells during Culture in vitro

Human embryonic fibroblasts （hEFs） can well maintain the pluripotency in human embryonic stem cells （hESs）. However, recent research and reports indicated that a few of hES cell lines acquired genomic alteration during long-term culture of hES cells in vitro. This will directly restrict the therapy use of hES cells. Wnts are secreted lipid-modified signaling proteins that influence multiple processes ranging from cell proliferation to stem cell loss. Activation of Wnt signaling in many tissues has also been associated with cancer. Unchecked Wnt signaling and loss of cadherin expression can promote tumorigenesis. In this study, we found the caryotype of one hES cell line chHES-3 changed with duplication of 1 p32-1p36 area after 34 passages. The results of RT-PCR indicated Wnt7a was expressed in hEFs after culture normal karyotype hES cells, but not expressed in control and abnormal karyotype hES cells. Wnt3 was expressed in hEFs after culture abnormal karyotype hES cells, not expressed in control and normal karyotype hES cells. Wnt3, Wnt9a and WntlOb were detected weakly expression in normal hES cells, but higher in abnormal hES cells. At the same time, Wnt3a, Wnt4, Wnt5b, Wnt7a, Wnt8b and Wnt11 were expressed and E-cadherin was not tested in abnormal hES cells compared with normal hES cells. All that indicated Wnt7a was need for culture normal karyotype hES cells and Wnt3 was need for culture abnormal karyotype hES cells on hEFs. Wnt3, Wnt9a and WntlOb high expression in hES cells and absence of E-cadherin may cause hES cells karyotype change.

Differential Expression of Bt Protein in Transgenic Bt Cotton

[Objective] The paper was to study the temporal and spatial dynamics of Bt protein expression in transgenic Bt cotton and to determine the inner relationship of Bt protein expression and transgenic Bt cotton. [Method] With transgenic cotton cultivar（ GK19） as the test material,Bt protein contents in different organs,main stem functional leaves at different growth stages and different positions of main stem leaves at different growth stages were studied by enzyme-linked immunosorbent assay. [Result] There were differences in Bt protein content among different organs of transgenic Bt cotton; the Bt protein content of leaves at seedling stage was the highest,followed by flowers,bubs and bolls,and those of roots and stems were relatively low. The Bt protein content of main stem function leaves gradually decreased with the progressing development. There were great differences in Bt protein content among different positions of main stem leaves at different growth stages; the Bt protein content of the 1^（st）-7^（th） top leaves at seedling stage and full budding stage gradually decreased,while those at full flowering stage and full bolling stage first slowly increased then gradually stabilized. [Conclusion] Bt protein expression was found in all organs of transgenic cotton at all growth stages,and the expression level presented temporal and spatial dynamics.

Identification of cellular genes showing differential expression associated with hepatitis B virus infection

AIM: To investigate the impact of hepatitis B virus (HBV) infection on cellular gene expression, by conducting both in vitro and in vivo studies. METHODS: Knockdown of HBV was targeted by stable expression of short hairpin RNA (shRNA) in huH-1 cells. Cellular gene expression was compared using a human 30K cDNA microarray in the cells and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) (qRT-PCR) in the cells, hepatocellular carcinoma (HCC) and surrounding non-cancerous liver tissues (SL). RESULTS: The expressions of HBsAg and HBx protein were markedly suppressed in the cells and in HBx transgenic mouse liver, respectively, after introduction of shRNA. Of the 30K genes studied, 135 and 103 genes were identified as being down- and up-regulated, respectively, by at least twofold in the knockdown cells. Functional annotation revealed that 85 and 62 genes were classified into four up-regulated and five down-regulated functional categories, respectively. When gene expression levels were compared between HCC and SL, eight candidate genes that were confirmed to be up- or down-regulated in the knockdown cells by both microarray and qRT-PCR analyses were not expressed as expected from HBV reduction in HCC, but had similar expression patterns in HBV- and hepatitis C virus-associated cases. In contrast, among the eight genes, only APM2 was constantly repressed in HBV non-associated tissues irrespective of HCC or SL. CONCLUSION: The signature of cellular gene expression should provide new information regarding the pathophysiological mechanisms of persistent hepatitis and hepatocarcinogenesis that are associated with HBV infection.

Differential Expression of Salinity Resistance Gene on Cotton

Salinity resistance and differential gene expression associated with salinity in cotton germplasm were studied,because of the large scale area of salinity in China,and its significant negative effects

Decoding Retinoblastoma: Differential Gene Expression

Background: Retinoblastoma, the most common intraocular pediatric cancer, presents complexities in its genetic landscape that necessitate a deeper understanding for improved therapeutic interventions. This study leverages computational tools to dissect the differential gene expression profiles in retinoblastoma. Methods: Employing an in silico approach, we analyzed gene expression data from public repositories by applying rigorous statistical models, including limma and de seq 2, for identifying differentially expressed genes DEGs. Our findings were validated through cross-referencing with independent datasets and existing literature. We further employed functional annotation and pathway analysis to elucidate the biological significance of these DEGs. Results: Our computational analysis confirmed the dysregulation of key retinoblastoma-associated genes. In comparison to normal retinal tissue, RB1 exhibited a 2.5-fold increase in expression (adjusted p Conclusions: Our analysis reinforces the critical genetic alterations known in retinoblastoma and unveils new avenues for research into the disease’s molecular basis. The discovery of chemoresistance markers and immune-related genes opens potential pathways for personalized treatment strategies. The study’s outcomes emphasize the power of in silico analyses in unraveling complex cancer genomics.