Display Reverse Transcriptase-polymerase Chain Reaction Research Articles

Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) for Grey Oyster Mushroom Samples Grown with Acoustic Sound Treatment

Grey oyster mushroom is the most often farmed and marketed type of mushroom for household consumption in Malaysia. The mushroom cultivation takes a long time due to its complicated mycelium growth. Several treatments were introduced such as acoustic sound treatment to increase the growth rate and quality of the crop, but there is no in-depth study regarding the genetic expression of the genes affected. This study aims to evaluate the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) for grey oyster mushroom samples grown with acoustic sound treatment. The mushroom was subjected to the treatment and the RNA was extracted from the mushroom samples and converted into cDNA before undergoing DDRT-PCR. Ten Differential Expressed Transcripts (DETs) that were successfully identified based on the differences between the intensity and absence of amplicons were sent for gene sequencing and BLAST through the NCBI database to obtain relevant results regarding the possible gene annotation. Seven out of ten DETs hit potential genes encoding for housekeeping and structural and development functions. The results showed that acoustic sound treatment did affect the expression of certain genes differently as captured by DDRT-PCR analyses and offers new ideas for the development of ecological agriculture.

Differential display analysis of cDNA fragments potentially involved in Nostoc flagelliforme response to osmotic stress

Nostoc flagelliforme is a terrestrial, highly drought-tolerant cyanobacterium. However, the molecular mechanism of its drought adaptation still remains little known. In this paper, we attempted to isolate those osmotic stress-regulated cDNA fragments by traditional differential display reverse transcriptase–polymerase chain reaction (DDRT-PCR). The PCR primers were chosen and evaluated from the reported HIP1D sequence-based primers and Ea/Es primers. The physiologically recovered samples were treated with 0.7 M sorbitol for 3 h. A total of 21 cDNA fragments were obtained by DDRT-PCR, and among them, 16 are up-regulated by osmotic stress. BlastX analyses showed that these up-regulated cDNA fragments are most related to ATPases, methyltransferases, transporters, glutathione synthase, signal transduction-associated components, and other unknown proteins. They are potentially important genes for N. flagelliforme to cope with drought stress since they are distinctly up-regulated by osmotic stress. In addition, five cDNA fragments are down-regulated by osmotic stress, such as the cDNA fragment of PSII protein. They may represent those genes involved in normal physiological processes and thus were inhibited by the osmotic stress. Taken together, the interpretation of the functional clues of these osmotic stress-responsive cDNA fragments has provided us an insight into the molecular mechanism of N. flagelliforme coping with the early phase of drought stress.

Presenilin enhancer-2 (PSENEN), a component of the γ-secretase complex, is involved in adipocyte differentiation

This study was conducted to identify genes expressed during adipocyte differentiation of bovine intramuscular fibroblast-like cells using differential display reverse-transcriptase polymerase chain reaction. The presenilin enhancer-2 (PSENEN) gene was found to be down-regulated during adipocyte differentiation of bovine intramuscular fibroblast-like cells. The ectopic expression of bovine PSENEN in 3T3-L1 reduced adipogenesis and the inhibition of endogenous PSENEN by siRNA induced adipogenesis on d 4 of adipocyte differentiation of 3T3-L1 cells. Interestingly, the expression of γ-secretase complex gene-related Notch signaling was decreased at d 2 and d 4 during adipocyte differentiation. In addition, expression of the Notch-signaling genes (Notch-1, Hes-1, Pref-1, adipsin) was regulated during adipocyte differentiation by regulation of PSENEN expression. These results suggest that PSENEN plays an important role in adipocyte differentiation and that Notch signaling is involved in adipogenesis.

Differentiation Associated Changes in Gene Expression Profiles of Interstitial Cystitis and Control Urothelial Cells

We evaluated gene expression profiles after inducing differentiation in cultured interstitial cystitis and control urothelial cells. Bladder biopsies were taken from patients with interstitial cystitis and controls, that is women undergoing surgery for stress incontinence. Primary cultures were grown in keratinocyte growth medium with supplements. To induce differentiation in some plates the medium was changed to Dulbecco's modified Eagle's minimal essential medium-F12 (Media Tech, Herndon, Virginia) with supplements. RNA was analyzed with Affymetrix(R) chips. Three patients with nonulcerative interstitial cystitis were compared with 3 controls. After inducing differentiation 302 genes with a described function were altered at least 3-fold in interstitial cystitis and control cells (p <0.01). Functions of the 162 up-regulated genes included cell adhesion (eg claudins, occludin and cingulin), urothelial differentiation, the retinoic acid pathway and keratinocyte differentiation (eg skin cornified envelope components). The 140 down-regulated transcripts included genes associated with basal urothelium (eg p63, integrins beta4, alpha5 and alpha6, basonuclin 1 and extracellular matrix components), vimentin, metallothioneins, and members of the Wnt and Notch pathways. When comparing interstitial cystitis control cells after differentiation, only 7 genes with a described function were altered at least 3-fold (p <0.01). PI3, SERPINB4, CYP2C8, EFEMP2 and SEPP1 were decreased, and AKR1C2 and MKNK1 were increased in interstitial cystitis cases. Differentiation associated changes occurred in interstitial cystitis and control cells. Comparing interstitial cystitis vs control cases revealed few differences. This study may have included patients with interstitial cystitis and minimal urothelial deficiency, and/or we may have selected cells that were most robust in culture. Also, the abnormal urothelium in interstitial cystitis cases may be due to post-translational changes and/or to the bladder environment.

Molecular characterization of an expressed sequence tag representing the cuticle-degrading serine protease gene (PII) from the nematophagous fungus Arthrobotrys oviformis by differential display technology

The technology of mRNA-based differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to detect a 246-bp differentially expressed fragment from the nematophagous fungus Arthrobotrys oviformis when young mycelia were induced with the round worm Haemonchus contortus. The fragment was converted into an expressed sequence tag (EST) through characterization at the molecular level. Homology search indicated that the differentially expressed fragment originated from the cuticle-degrading serine protease gene, which has been previously reported to play a role in nematophagous activity in A. oligospora, Dactylaria parvispora, A. musiformis, and other potential anti-fungal biological control agents. Several single nucleotide polymorphisms found to represent both synonymous as well as non-synonymous mutations within this short sequence stretch of 246 bp suggested genetic variability within the gene in this group of nematode-trapping fungi. The cloned EST fragment has potential for use as a hybridization probe for searching full-length gene from an appropriate cDNA library of this and related fungi. This is the first report of the identification of an EST representing the cuticle-degrading serine protease gene from A. oviformis using the technique of DDRT-PCR.

Tolerance of submergence in rice: gene studies using differential display technique

AbstractGene expression profiles between submergence rice (Oryza sativa ssp. indica) varieties, tolerant FR13A and sensitive IR39595-503-2-1-2, under submergence stress were identified using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). A total of 1428 bands were amplified with 40 pairs of primers, of which 102 were significantly different between the two lines. The differential display ratio was 7.1%. Among 42 differential display bands derived from the submergence tolerant variety, the expression of seven fragments was confirmed by Northern blot analysis. The analysis of their sequences indicated that four of them showed high homology with genes related to a water stress response: genes encoding ATP-binding protein, isocitrate dehydrogenase, NADH dehydrogenase and terminal acetyltransferase, respectively. The remaining three fragments were novel cDNA fragments.

Differential display fingerprints: new approach to characterize smooth muscle cells and human coronary atherectomy tissues

Smooth muscle cells build up the normal media and stabilize atherosclerotic lesions whereas an inflammatory component is determinant for unstable angina. Smooth muscle cells, currently identified by alpha-actin, present a phenotypic heterogeneity and alpha-actin can be reduced in pathology. We tried to characterize vascular cell types, particularly smooth muscle cells, and coronary atherosclerotic tissues, by random genes expression fingerprints. Expression fingerprints (cDNA electrophoresis) were performed by differential display reverse transcriptase-polymerase chain reaction. Variability of fingerprints was studied for a panel of arterial muscle cell phenotypes and comparisons were made with fingerprints from other cell types (endothelial cells and macrophages). The technique was then applied to human coronary atherectomy samples compared to control human arterial (mammary) smooth muscle. Arterial smooth muscle cells fingerprints were overall similar whatever the cell phenotype (native contractile, dedifferentiated in culture or epithelioid). Moreover, with two primer pairs, the muscular fingerprints markedly differed from the endothelial and the monocytic fingerprints. Application of differential display to coronary atherectomy samples was feasible. Interestingly, the pathological tissues exhibited either smooth muscle-like or smooth muscle-divergent fingerprints. Smooth muscle cells and inflammatory cells exhibited distinct differential display fingerprint patterns. Thus, a simple expression profile of arbitrary genes provides a molecular bar code tool (pattern signature) useful to characterize vascular cell cultures or tissues. The present work proposes a method to analyze coronary atherectomy samples which estimates their whole quality, muscular versus non muscular (inflammatory), this is of interest for clinical research.

Mining of Biomarker Genes from Expressed Sequence Tags and Differential Display Reverse Transcriptase-Polymerase Chain Reaction in the Self-fertilizing Fish, Kryptolebias marmoratus and Their Expression Patterns in Response to Exposure to an Endocrine-disrupting Alkylphenol, Bisphenol A

Mining of Biomarker Genes from Expressed Sequence Tags and Differential Display Reverse Transcriptase-Polymerase Chain Reaction in the Self-fertilizing Fish, Kryptolebias marmoratus and Their Expression Patterns in Response to Exposure to an Endocrine-disrupting Alkylphenol, Bisphenol A

Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion

Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.

Novel Urothelium Specific Gene Expression Identified by Differential Display Reverse Transcriptase-Polymerase Chain Reaction

Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

Investigation of alpha nascent polypeptide-associated complex functions in a human CD8(+) T cell ex vivo expansion model using antisense oligonucleotides.

In order to determine molecules involved in the differentiation and proliferation of human CD8(+) cells, two ex vivo expansion models were established: coculture of freshly purified human CD8(+) cells with irradiated autologous feeders (AF) or stimulation with anti-CD3. Two different proliferation kinetics of CD8(+) cells and expression patterns of CD57 were observed between these conditions. Differential display reverse transcriptase-polymerase chain reaction was applied to investigate the differential expression of mRNA species between CD8(+) CD57(+) and CD8(+) CD57(-) populations. A differentially expressed RNA species called alpha nascent polypeptide associated complex (alpha NAC) was found at a higher level in CD8(+) CD57(-) cells than in CD8(+) CD57(+) cells. In the presence of AF, the expression of alpha NAC was reduced on culturing whilst proliferation increased. Similarly, in cultures stimulated with anti-CD3, alpha NAC reverted to its inactive form and differentiation and proliferation increased. Using a phosphorothioate-modified oligodeoxynucleotide antisense directed specifically against alpha NAC mRNA, protein expression was inhibited and increased CD8(+) cell proliferation and CD25 expression were observed irrespective of the culture conditions. This suggests that alpha NAC protein is antiproliferative molecule. This is the first description of the function of the alpha NAC protein in human CD8(+) T cells.

Screening for differential gene expression during the development of form-deprivation myopia in the chicken.

To use the technique of differential gene display to analyze changes in gene expression that occur during the development of and recovery from form-deprivation myopia. The differential display-reverse transcriptase-polymerase chain reaction technique was used to detect cDNAs that are differentially expressed after 24 h (including 12 h in the light) after fitting with a diffuser to induce form-deprivation myopia. Messenger RNA levels were determined by quantitative Northern blotting in retinas after 11 days of form deprivation or in retinas where the diffusers had been removed the previous day. Twenty-six differentially expressed genes were processed in our initial screen. Two of these, alphaB-crystallin and retinoic acid receptor-alpha, were studied further. Levels of alphaB-crystallin mRNA were increased on day 11 in retinas from form-deprived eyes relative to eyes of control chickens and were reduced to below those levels within 6 to 12 h after removal of the diffusers. Levels of retinoic acid receptor-alpha mRNA showed similar changes, except that after removal of the diffusers, the levels further increased. The technique of differential gene display can be used to detect changes in gene expression during the regulation of eye growth. The response of alphaB-crystallin is particularly interesting because expression increases when eye growth is high and decreases when eye growth slows.

RNA extraction from cartilage.

The direct isolation of RNA from cartilage has often proved difficult owing to a number of factors. Cartilage has a low cell content and contains an extracellular matrix rich in proteoglycans, which copurify with the RNA as they are large and negatively charged macromolecules. In our laboratory, we are interested in searching for genes differentially expressed in chondrocytes in diverse in vivo situations, for instance during maturation of chondrocytes in the growth plate or during cartilage degeneration. We found that treatment by proteinase K in 1 M guanidinium isothiocyanate prior to cesium trifluoroacetate ultracentrifugation was crucial to increase the yield and purity of RNA extracted from cartilage matrix. This protocol indeed led to reproducible patterns of differential display reverse transcriptase-polymerase chain reaction (RT-PCR) and should be useful for identifying genes differentially expressed by chondrocytes in situ.

Differentially expressed genes and estrogen receptor status in breast cancer

There is a large and increasing body of experimental and clinical data supporting the involvement of estrogen on the proliferation of hormone-dependent breast tumors. Estrogen acts via its receptor (ER) stimulating cellular proliferation. ER and progesterone receptor (PR), which is regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. The aim of the present study was the identification of tumor-associated genes differentially expressed in breast tumors regarding the presence or absence of ER and PR. Using the technique of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we have isolated and cloned 127 cDNA fragments that showed differential expression in either ER+/PR+ or ER-/PR- breast tumors. Sequencing analysis of these clones revealed that 119 cDNAs had homology with known sequences in the National Center of Biotechnology Information (NCBI) and 8 were novel, showing no homology to known genes. Among these differentially expressed transcripts are metabolic enzymes, ribosomal proteins, transcription factors, hypothetical proteins, cell cycle regulators, cytoskelectum related genes, cell adhesion and motility genes. Differences in gene expression profiles are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast tumors.

MtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells.

To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

Identification and characterization of a phytoestrogen-specific gene from the MCF-7 human breast cancer cell

Phytoestrogens are a group of compounds present in human diet that display estrogenic-like properties. Several studies have demonstrated that populations who consume large quantities of phytoestrogens have a reduced risk of estrogen-dependent cancers. Although it has been shown that certain phytoestrogens modulate estrogen action, their biological role in cancer reduction remains unclear. Through the use of differential display reverse transcriptase–polymerase chain reaction and representational difference analysis of cDNA, we have identified several phytoestrogen-responsive genes from the human breast cancer cell MCF-7. Two of these genes, PE-13.1 and pRDA-D, have been characterized in greater detail in this study. These genes were not previously known to be regulated by phytoestrogen or estradiol. PE-13.1 is a novel gene that specifies the coding of a 1.10-kb mRNA transcript. Northern blot analysis confirmed that the PE-13.1 transcript is up-regulated by phytoestrogens (Genistein, sevenfold; Zearalenone, twofold) and is nonresponsive to estradiol. Conversely, the pRDA-D transcript was down-regulated by both phytoestrogens and estradiol. The antiestrogen ICI-182,780 inhibits the expression of PE-13.1 and reverses the inhibition of pRDA-D expression induced by phytoestrogens and estradiol. Analysis of the tissue distribution of PE-13.1 transcript by RNA blot reveals that this transcript is expressed in both normal and tumor tissues. This report demonstrates for the first time the presence of two phytoestrogen-responsive genes that may be used as molecular markers in understanding the role dietary estrogen plays in cancer prevention.

Temporal regulation of gene expression during the expected window of receptivity in the rhesus monkey endometrium.

Progesterone has been shown to regulate a number of genes and gene networks in the primate endometrium. This action of progesterone is essential to provide an appropriate milieu for embryo-endometrial communication that can lead to implantation and the successful initiation of pregnancy. A temporal regulation of endometrial genes is most likely required to achieve an appropriate state of receptivity in the primate endometrium. Using simulated menstrual cycles in the rhesus monkey, endometrial tissue was harvested at days that encompass the expected window of receptivity (4-10 days after the estradiol surge) and subsequently converted to cycle day-specific cDNA populations. Using differential display reverse transcriptase-polymerase chain reaction, 12 cDNA fragments were isolated and sequenced whose mRNA levels were elevated during this time frame. The temporal expression patterns of these mRNAs were confirmed by semiquantitative polymerase chain reaction. Two of these fragments exhibited high homology to previously characterized human genes: 1) secretory leukocyte protease inhibitor, also known as antileukoprotease, an endometrial neutrophil elastase inhibitor with antibacterial and anti-inflammatory properties; and 2) syncytin, also known as endogenous retrovirus W envelope protein, a highly fusogenic membrane glycoprotein that induces formation of giant syncytia and is believed to be important in decidual and placental development. The temporal regulation of these genes by progesterone supports their likely role in the orchestration of molecular and cellular events that are required to achieve a state of receptivity in the primate endometrium.

Identification of an encystation-specific transcription factor, Myb protein in Giardia lamblia.

The life cycle of Giardia lamblia contains two differentiation processes, encystation and excystation. We performed an experiment to identify the genes induced during encystation using the differential display reverse transcriptase-polymerase chain reaction. Three of twelve isolated cDNA clones that showed increased transcription during encystation were identified to be of the myb2, which encodes a well-known transcriptional factor involved in cellular development and differentiation. The amino acid sequences of the Myb2 protein deduced from the isolated gene revealed that this Myb2 has a DNA binding domain comprising two imperfect repeats at its carboxyl-terminus. The nuclear localization of Myb2 protein during encystation was observed in vivo by expressing a Myb2-GFP fusion protein. In a random site selection experiment, the oligonucleotides bound by rMyb2 contained a conserved sequence of GTTT(G/T)(G/T). Two promoters of the encystation-induced genes, myb2, and cwp1, were also found to bind to rMyb2, whereas gap1, one of the constitutive genes did not.

The ribosomal small‐subunit protein S28 gene from Helianthus annuus (Asteraceae) is down‐regulated in response to drought, high salinity, and abscisic acid

A partial cDNA for the ribosomal S28 gene from sunflower was initially cloned and identified to be down-regulated by high salinity, using differential display reverse transcriptase-polymerase chain reaction (PCR). Using this sequence, a 502-base pair (bp) full-length cDNA was cloned by rapid amplification of cDNA ends. This cDNA (designated Ha-RPS28) encodes a protein component of the small subunit of cytoplasmic ribosomes. The predicted 65 amino acid residue sequence of Ha-RPS28, with an estimated molecular mass of 7.5 kD, has 92, 89, and 86% identity with the S28 ribosomal proteins from peach, maize, and Arabidopsis, respectively. Ha-RPS28 was expressed in all organs examined, and the highest level was detected in fully expanded leaves. Furthermore, expression of Ha-RPS28 was down-regulated in both seedling roots and shoots in response to drought, high salinity, or abscisic acid.

Expression of testicular germ cell genes identified by differential display analysis.

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations.