Fluorescence differential display (FDD) technique was used to identify genes that are specifically or preferentially expressed in different developmental stages of cotton fiber cells. One hundred and nine differentially displayed cDNA fragments were isolated using 9, 21 and 27 DPA (days postanthesis) fibers as experimental materials. By a combination of two rounds of reverse Northern hybridization and Northern blot analyses, a number of such cDNA fragments were proved to represent fiber-specific/preferential genes. Sequencing determination and database searching indicated that most of these genes are novel. This work is an important step towards cloning the full-length cDNAs and characterizing the cellular functions of aforementioned genes in fiber development.
Blotting, Northern ; Cotton Fiber ; Fluorescence ; Gene Expression Profiling ; methods ; Gossypium ; genetics ; growth & development ; Polymerase Chain Reaction

Microarray analyses of gene expression are widely used, but reports of the same analyses by different groups give widely divergent results, and raise questions regarding reproducibility and reliability. We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes. These reports show substantial differences in their results. In this article, we review the methodology, results, and potential causes of differences in these applications of microarrays. Finally, we suggest practices to improve the reliability and reproducibility of microarray experiments.
Animals ; Blotting, Northern ; Gene Expression Regulation ; Genomics ; methods ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Reproducibility of Results ; Tretinoin ; chemistry

Apoptosis was induced by taxol treatment in suspension cultures of Taxus cuspidata cells. Differential display technique was used to investigate the induced-gene expression between the taxol-induced T. cuspidata cells and normal control. Eight different expressed cDNA fragments were cloned and sequenced. These differential expressed fragments were further confirmed by Northern blotting hybridization with their original total RNAs. The result showed that three of the cDNA fragments were from control RNA and five of those were from taxol-induced T. cuspidata cells. The homology of the sequences revealed that one of the clones had 86% homology with ABA-responsive protein gene sequence in Arabidopsis thaliana, two of the clones had 50% homology with endochitinase precursor in tomato and the other 5 clones, which might be new gene fragments, had no significant homology with the known gene sequences in GenBank/EMBL/DDBJ.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; Base Sequence ; Blotting, Northern ; methods ; Cells, Cultured ; DNA, Plant ; Molecular Sequence Data ; Paclitaxel ; pharmacology ; Polymerase Chain Reaction ; methods ; Taxus ; cytology ; genetics

BACKGROUND: Diabetes mellitus is a heterogeneous group of disorders characterized by high serum glucose levels and by disturbances of carbohydrate and lipid metabolism. There are many cutaneous signs of this common endocrinopathy, such as nercobiosis lipoidica diabeticorum, diabetic bullosis, shin spot, diabetic pruritus, etc. OBJECTIVE: In this study, we investigated whether extracellular matrix gene expression in cultured skin fibroblast is influenced by insulin and Insulin-like growth factor-I(IGF-I). METHOD: Total RNA was isolated from insulin or IGF-I treated human skin fibroblasts. The Northern blot and slot-blot hybridization were then conducted. RESULTS: The mRNA levels of pro α1(I) collagen, pro α1(I11) collagen, fibronectin in insulin and IGF-I treated normal skin fibroblasts increased compared with untreated normal skin fibroblasts. CONCLUSION: Our results show that insulin and IGF-I stimulate collagen formation in normal skin fibroblast at physiological concentrations. Therefore, these demonstrate that insulin can modulate the expression of extracellular matrix gene.
Blood Glucose ; Blotting, Northern ; Collagen ; Diabetes Mellitus ; Extracellular Matrix* ; Fibroblasts* ; Fibronectins ; Gene Expression ; Humans ; Insulin* ; Insulin-Like Growth Factor I ; Lipid Metabolism ; Methods ; Pruritus ; RNA ; RNA, Messenger ; Skin*

OBJECTIVETo identify hemin-induced gene expression in K562 cells.

METHODSPoly A(+) RNAs were isolated from hemin-induced (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNAs were synthesized by reverse transcription. The forward subtracted cDNA library was constructed by using suppression subtractive hybridization (SSH) techniques. The recombinant plasmids were extracted and the positive clones were identified by EcoR I digestion after the amplification and screening of the library. The inserts were amplified by PCR. The upregulated cDNA transcripts were identified by reverse dot blot hybridization, DNA sequencing and homology analysis with GenBank database "blast" respectively.

RESULTSFifteen upregulated clones were identified and most of them were homologous to the mRNA sequences of protein with known function, including globin epsilon1, glutathione S-transferase like glutathione transferase Omega (GSTTLp28), selenoprotein X1 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin light polypeptide, globin A gamma, RAD 51 homolog C(RAD51C), ferritin heavy polypeptide, X-box binding protein (XBP1). A part of the hemin-induced cDNA clones exhibited sequence similarities to that of the GenBank registered mRNA with unknown function of their expressed proteins, including the cDNA clones of DKFZp434I116, hypothetical protein HSPC014 and NOL1R2 proteins.

CONCLUSIONSHemin mainly induces the genes expression related to erythroid differentiation, protein synthesis and metabolism in K562 cell. There results provide comprehensive information useful for the differential gene expression in hemin-induced erythroid differentiation and for further function study of genes involved in hematopoiesis.

Blotting, Northern ; DNA, Complementary ; drug effects ; genetics ; Gene Expression ; drug effects ; Hemin ; pharmacology ; Humans ; K562 Cells ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction

Biliary atresia is a progressive obliterative cholangiopathy, but the etiology of this disorder remains uncertain. Identifying genes specifically expressed in biliary atresia and analyzing the pattern of expression may lead to a better understanding of the pathogenesis. Liver tissues were taken from a recipient with biliary atresia and a normal donor during liver transplantation. Total RNA was extracted from each sample and reversely transcribed to cDNA. Then radiolabeled cDNA probe pools were made by random primed DNA labeling method and used for screening of differentially expressed genes by hybridizing with expressed sequence tags (EST) dot blot panel. Northern blot hybridization was done to confirm that these genes are also differentially expressed in other liver tissues. Among 1,730 EST clones, 26 cDNA clones were significantly overexpressed in biliary cirrhosis, while 2 clones were significantly decreased in biliary atresia. By Northern blot hybridization, the results of tissue inhibitor of metalloproteinase (TIMP)-1 and IGFBP-2 were well correlated with differential EST screening (DES). This study identified the pattern of differentially expressed genes in the biliary cirrhosis due to biliary atresia using DES technique.
Biliary Atresia/*genetics ; Blotting, Northern ; Gene Expression Profiling/*methods ; Gene Library ; Human ; Insulin-Like Growth Factor-Binding Protein 2/genetics ; Tissue-Inhibitor of Metalloproteinase-1/genetics

A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeastmalt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.
Agar ; Agaricales* ; Blotting, Northern ; Clone Cells ; Culture Media ; DNA, Complementary ; Electrophoresis ; Fruit ; Fungal Viruses* ; Methods ; Pleurotus* ; RNA Replicase ; RNA, Double-Stranded ; Weights and Measures

Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.
Blotting, Northern ; methods ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Library ; Gene Regulatory Networks ; Humans ; Nucleic Acid Hybridization ; methods ; Space Flight ; Up-Regulation ; Uterine Cervical Neoplasms ; genetics ; pathology

BACKGROUND: bFGF, a member of the fibroblast growth factor family, potently induces vascular smooth muscle cell proliferation and decreased synthesis of the collagens. OBJECTIVE: For further investigation of the effect of bFGF on extracellular matrix homeostasis in the skin, we evaluated the expression of type I and type VII collagen gene at the transcriptional levels. METHOD: We examined that recombinant human bFGF affects the expression of genes involved in ECM synthesis and remodeling in human dermal fibroblasts cultures as judged by Northern blot analysis. RESULTS: The steady state levels of type I and VII collagen gene mRNA were decreased with age dependent pattern up to 0.13 and 0.44 folds respectively. The transcriptional levels of type I collagen mRNA were increased by TGF-B, treatment but markedly decreased by bFGF as well as TNF-a. But there were no synergistic effects bFGF and TNF-a on type I collagen gene expression. The levels of type VII collagen gene expression were increased by both bFGF and TGF-B,. The TNF-a showed slightly antagnostic effects on type VII collagen gene expression. CONCLUSION: The type I and VII collagen gene expression in dermal fibroblasts is clearly subjected to modulation by the cytokines including bFGF with uncoordinate regulatory pathway. In addition to its function of vascular proliferation, bFGF also may play a major role in physiologic skin condition and in repair process such as formation of a stable dermoepidermal junction during skin wound healing.
Blotting, Northern ; Cell Proliferation ; Collagen Type I ; Collagen Type VII ; Collagen* ; Cytokines ; Extracellular Matrix ; Fibroblast Growth Factors ; Fibroblasts* ; Gene Expression* ; Homeostasis ; Humans ; Methods ; Muscle, Smooth, Vascular ; RNA, Messenger ; Skin ; Wound Healing

OBJECTIVETo clone and screen genes related to multidrug resistance (MDR) in leukemia.

METHODSSuppression subtractive hybridization (SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver). Reverse Northern dot blot was carried out to further screen the subtracted cDNA library. The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank. RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes.

RESULTSEleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp) gene, NADH dehydrogenase subunit 2 (ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer.

CONCLUSIONSeveral genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.

Blotting, Northern ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Library ; Genes, MDR ; genetics ; Humans ; K562 Cells ; Leukemia ; genetics ; NADH Dehydrogenase ; genetics ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosomal Proteins ; genetics