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At least 1,117 records · Page 62Linked to original sources
PubMed2006

Uroepithelial cells can directly respond to Mycobacterium bovis bacillus Calmette-Guérin through Toll-like receptor signalling.

OBJECTIVE: To investigate, in a human urinary tract cell line, the interaction of Toll-like receptor (TLR) signals with cytoplasmic adapter proteins MyD88 and bacillus Calmette-Guérin (BCG), and evaluate the epithelial cytokine response to BCG infection. Intravesical BCG therapy is effective against carcinoma in situ and as prophylaxis for recurrence, but although immunological mechanisms have been assumed, the mechanisms of the antitumour effects of BCG have not been completely elucidated. MATERIALS AND METHODS: The cell line was first screened for TLR expression by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from a human urinary cell line, Hu35E6E7, and cDNA synthesised. PCR was used to measure the gene expression of TLR-2, -3, -4, -5, -9, MyD88, MD-2, CD14 and interleukin-8 and -6. The Hu35E6E7 cell line was cultured in keratinocyte serum-free medium, and BCG was added to the cell culture. After Hu35E6E7 cells were stimulated by BCG for various periods, the total RNA of the cells was extracted. Quantitative real-time PCR was conducted for MyD88 using appropriate probes, and the expression of MyD88 analysed. The cell supernatant was collected, and the levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-2, -12, -4, -6, -10, -8 and -1beta were assayed using an enzyme-linked immunosorbent assay. RESULTS: Uroepithelial cells expressed TLR-2, -3, -4 and -9, and MyD88, MD2, CD14, interleukin-6 and -8 were also detected. At 3, 6, 9 and 12 h after adding BCG, quantitative PCR assay showed that the expression of MyD88 was maximal at 6 h. The presence of BCG stimulated the release only of interleukin-6 and -8 from Hu35E6E7 cells after 6 h. By contrast, interferon-gamma, tumour necrosis factor-alpha, interleukin-2, -12, -4, -10 and -1beta were not detected in the culture supernatant. CONCLUSION: These results show that uroepithelial cells, but not immune cells, responded directly to BCG through TLR signalling. Further investigation is needed to determine the role of cytokines released from uroepithelial cells after BCG infection.

Animals↗
PubMed1992

Esterase-1: developmental expression in the mouse and distribution of related proteins in other species.

Esterase 1 (Es-1) is a sexually dimorphic 65-kDa glycoprotein present in plasma and other murine tissues able to hydrolyze a variety of esters including fatty acid esters of estradiol. Like most other carboxylesterases, its function is unknown. To gain insight into the function of Es-1 and by analogy other carboxylesterases, we have examined the developmental regulation of Es-1 in the mouse and have looked for the presence of related proteins in the plasma of other species. Northern blot analysis of total RNA from the livers of mice of various ages using a 32P-labeled 470-bp Es-1 cDNA probe showed clear postpartum induction with no detectable Es-1 mRNA in fetal liver. Similarly, immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an affinity-purified rabbit antibody to Es-1 showed no cross-reacting proteins in the plasma until after birth. Northern blot analysis of total RNA from a variety of adult mouse tissues showed the presence of substantial levels of Es-1 mRNA only in liver with lower levels in kidney, testes, and ovaries. Liver mRNA and plasma protein levels rose in parallel attaining full adult levels between 15 and 20 days of age. When plasma proteins were electrophoresed on 7% polyacrylamide gels under nondenaturing conditions, the antibody to Es-1 recognized a low mobility protein in mouse, rat, human, baboon, guinea pig, bovine, horse, and canine but not in chicken plasma. Consistent with the immunoblotting results, the Es-1 cDNA probe hybridized to restriction fragments from human, monkey, rat, and rabbit as well as mouse genomic DNA but not from chicken DNA indicating conservation of the esterase (or esterase-like) gene in mammalian species. The low mobility antigens in mouse and human plasma appeared also to cross-react with antibodies to human thyroglobulin, although antibodies to human thyroglobulin did not appear to recognize Es-1 under these conditions.

Aging↗
PubMedDate not supplied

Ovine glucose transporter-1 and -3: cDNA partial sequences and developmental gene expression in the placenta.

Transplacental glucose transfer utilizes specific glucose transporter (GLUT) proteins. cDNAs encoding ovine placental GLUT1 and GLUT3 genes were isolated, cloned and sequenced and then used to investigate their developmental regulation in ovine placenta. A cDNA of approximately 2.2 kb was isolated from a Clontech lambda gt10 ovine adult liver cDNA library using a 436-bp rat GLUT1 cDNA probe. Sequence data obtained from this clone (1600 bp) demonstrated 97 per cent homology to nucleotides 477-2079 of bovine GLUT1. The deduced amino acid sequence of the ovine cDNA presented 99 per cent identity to amino acid 103-493 of bovine GLUT1, and 97-98 per cent identity to corresponding regions in human and rat GLUT1 deduced amino acid sequences. Reverse transcription-PCR (RT-PCR) was used to isolate an ovine cDNA fragment from placental total RNA. Forward and reverse primers (16 mer) were designed to amplify a predicted 483-bp fragment between the second transmembrane-spanning domain (M2) and intracellular loop of GLUT3. The deduced 161 amino acid sequence of the ovine cDNA demonstrated 84 and 88 per cent identity with murine and human GLUT3. These cDNAs were used to investigate the ontogeny of placental oGLUT1 and oGLUT3 gene expression by Northern analysis. Total RNA was extracted from ovine placenta at gestational days 45, 60, 90, 120 and 138 (n=6 per time point). Ovine GLUT1 gene expression increased significantly from days 45 to 60 (P<0.05), peaked at around day 120 of gestation, then decreased to about two-thirds of maximal levels by day 138 of gestation (term 147 +/- 2). Ovine GLUT3 gene expression increased throughout gestation with significant increases from days 45 to 60, 60 to 120 and 120 to 138 (P<0.05). This study confirms the presence of both GLUT1 and GLUT3 genes in the ovine placenta and demonstrates ontogenic regulation of gene expression. The difference in temporal gene expression between oGLUT1 and oGLUT3 suggests distinct roles for each transporter during development. The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank under accession numbers U89029 (oGLUT1) and U89030 (oGLUT3).

Amino Acid Sequence↗
PubMed1990

Transferrin-gene expression in the rat mammary gland. Independence of maternal iron status.

Transferrin mRNA concentrations were measured in total RNA isolated from liver and mammary tissue of lactating rats at different stages of lactation. The mammary transferrin mRNA concentration varied in a biphasic manner, increasing up to parturition and then decreasing to undetectable levels at days 5 and 10 of lactation before increasing again markedly in late lactation. The values obtained at day 20 of lactation were double those found in livers of lactating rats. The concentrations of total RNA and mRNA for alpha-casein and alpha-lactalbumin did not change between days 5 and 20 of lactation. Transferrin concentrations were measured in milk from rats fed on an iron-free, a control and an iron-supplemented diet. Although there was a 5-fold difference in the transferrin concentration of samples taken between day 5 and day 20 of lactation, the dietary treatments did not result in significant changes. Maternal serum transferrin concentrations were, however, elevated, and pup haemoglobin concentrations were suppressed for the rats receiving the iron-free diet, indicating an alteration of the iron status of these rats.

Animals↗
PubMed1985

Translatable mRNA for GP140 (a subunit of type VI collagen) is absent in SV40 transformed fibroblasts.

Production of GP140, a major component of the extracellular matrix of cultured fibroblasts, is markedly decreased in SV40 transformed cells as compared with normal cells (Carter, W. G., 1982, J. Biol. Chem., 257:13805-13815). To determine at what step the biosynthesis is inhibited, we compared the levels of functional mRNA for GP140 in normal and transformed fibroblasts. Translation of total RNA from W138 cells in a reticulocyte lysate, followed by immunoprecipitation with affinity-purified antibodies to GP140, yielded a single polypeptide with an Mr of 125,000. This polypeptide was identified as GP140 based on its immunoreactivity, collagenase sensitivity, and comigration on polyacrylamide gels with GP140 synthesized by cells in the presence of tunicamycin and 2,2'-bipyridyl. No cell-free synthesis of GP140 was observed with total RNA from SV40 transformed W138 cells, indicating that these cells contain very low levels of GP140-specific mRNA. The biosynthesis of GP140 might therefore be blocked at the transcriptional level.

Cell Transformation, Viral↗
PubMed1997

Differential expression of human ferritin H chain gene in immortal human breast epithelial MCF-10F cells.

Subtractive hybridization was used to isolate genes expressed uniquely in the immortalized human breast epithelial cell (HBEC) line MCF-10F and not in the mortal HBEC line S-130, from which MCF-10F cells were derived. We identified a 233-bp cDNA that was expressed in MCF-10F cells and not in their mortal counterpart S-130 cells. Sequence comparison with the GenBank database revealed that the cDNA was identical to the gene encoding human ferritin heavy H chain. Northern blot analysis using the isolated cDNA as a probe showed a differentially expressed 1.1-kb transcript of ferritin H in total RNA from the immortal MCF-10F cells, MCF-10F cells treated with the chemical carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene, and the breast cancer cell lines MCF-7, HBL-100, T-47D, and BT-20. No ferritin H transcript was detected in the mortal line S-130 or in other primary HBEC cultures. Increased levels of mRNA transcript signals were also detected in total RNA from breast cancer tissue samples. Tissue with ductal hyperplasia had higher expression levels than normal adjacent mammary tissue. In situ hybridization showed high levels of ferritin H transcript in mammary tissue areas with ductal hyperplasia, carcinoma in situ, and infiltrating ductal carcinoma. This is the first report of the differential expression and upregulation of human ferritin H chain gene in immortal HBECs. It may be an important factor in the process of immortalization, possibly an early stage of malignant transformation of HBECs, providing cells with iron necessary for growth and clonal expansion. Also, ferritin iron, once released, may increase the level of reactive iron, leading to an increase in oxygen free-radical generation, oxidative DNA damage, and mutation.

Base Sequence↗
PubMed2002

Magnetic bead RT-PCR: establishment of a new method for detecting circulating melanoma cells.

This study was performed to detect circulating melanoma cells in peripheral blood using a novel method based on magnetic-activated cell separation (MACS) followed by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase and MART-1 mRNA. Samples to be tested were enriched for tumour cells either by isolating melanoma cells using two anti-melanoma antibodies (MART-1 and HMB-45) or by CD45 depletion of the non-melanoma cell fraction. The tumour cell-enriched fractions were subjected to mRNA isolation using oligo-deoxythymidylate (oligo-dT) magnetic beads followed by a nested RT-PCR. Sensitivity was assessed by spiking experiments and compared with a commonly used total RNA isolation system previously established in our department. Positive isolation of melanoma cells showed insufficient sensitivity, whereas negative isolation by depletion of leukocytes showed a detection limit of at least one melanoma cell per millilitre of whole blood. In further experiments, the depletion assay was applied to 25 peripheral blood samples of melanoma patients. The preliminary data obtained from the new method indicate a comparable detection rate to the established total RNA extraction method. However, not all the results were concordant. Therefore, future experiments need to be performed with a statistically greater number of patients.

Adult↗
PubMed2004

[Clinical values of vascular endothelial growth factor expression and microvascular density analysis in liver cancer specimens].

OBJECTIVE: To explore the roles of vascular endothelial growth factor (VEGF) in microvessel angiogenesis, development and metastasis of hepatocellular carcinoma (HCC). METHODS: The cellular distributions of VEGF expression and microvascular density (MVD) in 36 HCCs were investigated, and the levels of total RNA and VEGF were detected in HCCs, Para cancerous, and distal cancerous tissues, respectively. RESULTS: The incidence of VEGF was 63.9% in 36 cases of HCCs, 78.3% in non-encapsulated HCCs, and 90.9% in HCCs with extrahepatic metastasis, respectively. The VEGF expression was tightly correlated with MVD (t=4.49, P<0.01). No significant difference was found between VEGF or MVD and tumor diameter or differentiation degree. The level of total RNA in HCCs was lower but the VEGF level significantly higher than those of Para cancerous or distal cancerous ones (q=6.10, P<0.01). CONCLUSION: The present data suggest that VEGF over expression and MVD abnormality are useful markers for vascular invasion and metastasis of liver tumors.

Adult↗
PubMed1997

Nutritional regulation of the glucose-6-phosphate dehydrogenase gene is mediated by a nuclear posttranscriptional mechanism.

Expression of the glucose-6-phosphate dehydrogenase (G6PD) gene is inhibited by addition of polyunsaturated fat to a high-carbohydrate diet and stimulated by feeding a high-carbohydrate diet to starved mice. The mechanism of this regulation is posttranscriptional. To define the regulated step, we measured the abundance of G6PD mRNA both in the nucleus and in total RNA. Feeding mice a high-fat diet results in a 70% or greater inhibition of nuclear precursor mRNA (pre-mRNA) and mature mRNA abundance. Amounts of both pre-mRNA and mature mRNA for G6PD are stimulated 13-fold or more by refeeding starved mice. Changes in amount of pre-mRNA for G6PD are of a similar magnitude and precede the changes in amount of mature mRNA for G6PD in total RNA. These changes in pre-mRNA abundance occur in the absence of observable changes in the rate of transport of mRNA from the nucleus to the cytoplasm, splicing of the pre-mRNA, or degradation at the 3'-end of the transcript. Despite large changes in pre-mRNA amount in mice fed a low-fat diet relative to mice fed a high-fat diet, the rate of change in the amount of pre-mRNA during the diurnal feeding cycle is not altered. Thus, expression of G6PD is regulated at an early step after transcription of the pre-mRNA. We suggest that pre-mRNA which enters the processing pathway is stable and can be processed and transported to the cytoplasm where it is translated.

Animals↗
PubMed1997

Polymerase chain reaction-based construction of cDNA libraries from minute amounts of third-stage and fourth- and fifth-stage larvae of Dictyocaulus viviparus.

A strategy is described for the amplification and cloning of cDNA from minute amounts of Dictyocaulus viviparus larvae. Initially, third-stage larvae (L3) were used to establish the procedure. Amplification of cDNA synthesized from approximately 400 ng total RNA from 5,000 L3 generated products that were more than 800 bp in length. The unidirectional cloning of amplified cDNA products led to the construction of a UNI ZAP c DNA library with 1 x 10(6) clones. Screening with a homologous oligo(dT)-primed digoxigenin-labeled cDNA probe as well as sequencing of seven randomly picked clones confirmed the successful cloning of lung-worm cDNA. Subsequently, approximately 600 ng total RNA was isolated and polymerase chain reaction (PCR) products of up to 2,400 bp were amplitied from 400 fourth- and fifth-stage larvae (L4/L5). Cloning of these products resulted in a L4/L5 cDNA library of D. viviparus consisting of 5 x 10(5) recombinant clones. In all, 11 clones were randomly picked and sequenced, all revealing typical mRNA/cDNA characteristics. Comparison of the predicted amino acid sequence of the 5' end of clone DvL5/7 revealed 100% homology with the actin gene of several other helminths.

Actins↗
PubMed2006

Distribution and abundance of nutrient transporter mRNA in the intestinal tract of the black bear, Ursus americanus.

End products of digestion are absorbed by the body through the action of transporter proteins expressed on the apical membrane of intestinal epithelial cells. We investigated the mRNA abundance and distribution of a peptide transporter (PepT1), a glucose transporter (SGLT1), two amino acid transporters (NBAT and b(o,+)AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears (Ursus americanus). Intestinal total RNA was isolated from 10 bears and abundance of PepT1, SGLT1, NBAT, b(o,+)AT, and APN mRNA were determined by Northern blots. Abundance of PepT1 (P<0.05), APN (P<0.05), and SGLT1 (P<0.0001) changed quadratically from the proximal to distal intestine with abundance being greatest in the midregion. Abundance of b(o,+)AT mRNA increased linearly (P<0.05) from the proximal to distal intestine. The number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. PepT1 mRNA was present at 10-fold or greater levels than amino acid transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute a major form in which amino acids are absorbed in the black bear. The abundance of NBAT and b(o,+)AT mRNA was greater towards the distal intestine, suggesting a role in salvaging unabsorbed amino acids.

Animals↗
PubMed1994

Increased mRNA for CD63 antigen in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.

To identify a set of mRNAs that increases with the progression of atherosclerosis, differential hybridization screening of a cDNA library, constructed from total RNA in aortae of Watanabe heritable hyperlipidemic (WHHL) rabbits, was carried out. Here we report that one of the positive clones encodes a rabbit homologue of human CD63 antigen. Nucleotide sequence analysis of the cDNA revealed the primary structure of rabbit lysosomal membrane antigen CD63. Analysis of the mRNA by in situ hybridization revealed that a significant amount of the mRNA is accumulated in atherosclerotic lesions of WHHL rabbits. Northern blot analysis of total RNA prepared from aortae of WHHL and normal rabbits indicates that expression of the mRNA increased with age in WHHL rabbits, suggesting that expression of the mRNA is correlated with the progression of atherosclerosis.

Aging↗
PubMed2006

[Production of specific CTL induced by exosomes derived from K562 cells].

The aim of this study was to investigate whether exosomes derived from K562 cells and human monocyte-derived dendritic cells (DCs) transfected with total RNA of K562 cells are capable of inducing antigen-specific cytotoxic T lymphocytes (CTL) responses in vitro. DCs were generated from peripheral blood mononuclear cells (PBMNC) of healthy volunteers in the presence of GM-CSF and IL-4, and then were transfected with K562 RNA by using DOTAP lipofection. Exosomes was extracted from the supernatant of DCs and K562 cells. The T cell were activated to be tumor specific CTL after DCs and exosomes were co-cultured with autologous T cells derived from healthy volunteers' PBMNC. The effect of CTL on K562 cells was detected by MTT assay. The results showed that treatment of T cells with exosomes derived from K562 cells or DCs transfected with total RNA of K562 cells could significantly promote their killing ability on K562 cells as compared with untreated T cells (P < 0.05). The killing ability of T cells treated with exosomes on K562 cells was stronger than on HL-60 cells (P < 0.05). It is concluded that the specific CTL immune response to leukemia cells can be induced by exosomes derived from K562 cells.

Dendritic Cells↗
PubMed2001

Effect of chronic denervation and denervation-reinnervation on cytoplasmic creatine kinase transcript accumulation.

The extensor digitorum longus (EDL) and soleus muscles of adult mice were chronically denervated or denervated and allowed to reinnervate. Muscles were evaluated 1, 5, 14, 21, and 52 days after sciaticectomy. In terms of weight loss, myofiber atrophy, degeneration, and fibrosis, the soleus muscle was more affected than the EDL by chronic denervation. Fifty-two days after chronic denervation, the number of molecules of MCK/ng total RNA in both muscles (determined with competitive PCR) decreased, with the soleus muscle being more affected. At that stage, BCK mRNA levels in the denervated soleus were unchanged, but they were increased (>50%) in the EDL. Reinnervation restored MCK transcript accumulation in the EDL, whereas, in the soleus MCK, transcripts exceeded control values by 57%, approaching levels in the reinnervated EDL. Despite restoration of MCK mRNA levels, the number of molecules of BCK mRNA/ng total RNA was four- to fivefold higher in reinnervated versus control muscles, suggesting that the genes encoding the CK mRNAs are not coordinately regulated in adult muscle. The role of denervation induced, fiber type changes in regulating CK mRNA accumulation has been evaluated. Electron microscopic analyses have established that fibrosis is not a factor that determines BCK mRNA levels in the chronically denervated or denervated-reinnervated muscles. CK isozyme analyses support the hypothesis that a greater proportion of BCK mRNA found in 52 day chronically denervated and denervated-reinnervated muscles is produced in myofibers vs. nonmuscle cells than in control muscles.

Animals↗
PubMed2002

Comparison of different labeling methods for two-channel high-density microarray experiments.

In this report we evaluate three methods for labeling nucleic acids to be hybridized to a cDNA microarray: direct labeling, indirect amino-allyl labeling, and the dendrimer labeling method (Genisphere). The dendrimer method requires the smallest quantity of sample, 2.5 microg of total RNA compared with 20 microg with the direct or indirect methods. Therefore, we wanted to know whether the performance of the dendrimer method is comparable to the other methods, or whether significant information is lost. Performance can be considered in terms of sensitivity, dynamic range, and reproducibility of the quantitative signals for gene intensity. We compared the three labeling methods by generating three sets of eight self-to-self hybridizations using the same total RNA sample in all cases ("replicate study"). In our analysis, we controlled for the effects of print-tip and background subtraction biases. We also performed a smaller study, namely, a dilution series study with five dilution points per labeling method, to evaluate one aspect of predictive ability. From the replicate study, the dendrimer method appeared to perform as well, and often better, with respect to reproducibility and ability to detect expression. However, in the dilution series study, this method was outperformed by the other two in terms of predictive ability and did not perform very well. These findings are helping to guide our decisions on what labeling method to use for subsequent studies, based on the purpose of a specific study and its limitations in terms of available material.

Fluorescent Dyes↗
PubMed2000

Expression of gonadotropin and activin receptor messenger ribonucleic acid in human ovarian epithelial neoplasms.

Activin receptors (ActRs) and gonadotropin receptor mRNA expression were investigated in 18 human ovarian epithelial neoplasms. Northern blot analysis showed the presence of 3.0-kb type Ia ActR, 6.0- and 3.0-kb type IIa ActR, and 5.0-kb type IIb ActR mRNA transcripts in total RNA prepared from the cancer tissues. One carcinoma showed two major transcripts of a follicle-stimulating hormone receptor (FSH-R) gene, 4.1 and 2.4 kb, whereas the other two carcinomas showed two major transcripts of the luteinizing hormone/human chorionic gonadotropin receptor (LH-R) gene, 5.4 and 2.4 kb. These results were further analyzed by studying the corresponding PCR-amplified FSH and LH-R cDNA obtained by reverse transcription of total RNA. Expression of FSH-R mRNA was confirmed in about half of the cancer tissues. The size of the FSH-R reverse transcription-PCR product was the same as in normal ovarian follicles. Similarly, expression of LH-R mRNA was also detected in about half of the cancers. Normal ovaries and cancer tissues were homogenized, and activin concentrations were measured in extracts. Activin levels in normal ovarian tissue were around 0.59 +/- 0.01 ng/mg protein (mean +/- SE; n = 5), and activin production was detected in every cancer tissue, except one--serous adenocarcinoma. The findings in this study demonstrated that activin and ActRs are present in and synthesized by human ovarian epithelial neoplasms. Thus, activin seems to be available as an autocrine/paracrine factor in epithelial neoplasms and may contribute to the expression of FSH-R, although the roles of activin and gonadotropin in tumorigenesis has yet to be defined.

Activin Receptors↗
PubMed2001

[Experimental study of in osteogenesis in duced by NIH-3T3 cells transfected with bone morphogenetic protein 3 gene].

OBJECTIVE: To investigate the gene expression, protein synthesis and inducing osteogenetic capacity of NIH-3T3 cells transfected with recombinant human bone morphogenetic protein 3 (rhBMP3) gene. METHODS: Gene recombination technique was used to clone the BMP3 cDNA into PcDNA3 vector directly, and to form a recombined BMP3-PcDNA3 cDNA, which was introduced into NIH-3T3 cells by means of lipofectamine transfer. The total RNA of transfected NIH-3T3 cells was then extracted to verify the mRNA expression of BMP3 using Northern blot method. BMP3 protein synthesis and AKP activity in the transfected cells was checked. Then, the NIH-3T3 cells transfected with BMP3 gene were injected into nude mouse muscle to evaluate the inducing osteogenesis in the muscle. RESULTS: BMP3 gene had been inserted into PcDNA3 vector successfully. There was BMP3 mRNA expression in the total RNA of NIH-3T3 cells with the peak level appearing in the 6th week after transfection, and BMP3 protein was found in NIH-3T3 cells, and the AKP level is much higher in the transfected NIH-3T3 cells than in non-transfected NIH-3T3 cells. Histologic examination revealed that there were a large number of chondrocytes and trabeculae seen in the nude mouse muscle 4 weeks after injection of the transfected NIH-3T3 cells. CONCLUSION: BMP3 is a protein that possesses osteogenetic effect. After transfection with BMP3-PCDNA3, BMP3 mRNA expression and protein formation can be found in NIH-3T3 cells, and the transfected NIH-3T3 cells have the effect of inducing osteogenesis in vivo.

3T3 Cells↗
PubMed2001

cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol.

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 microg of total RNA and 1-2 microg of mRNA is required for each hybridisation, which is equivalent to 50-100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 microg of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2-4 cell layers with a fresh weight of approximately 0.5 mg.

DNA, Complementary↗