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PubMed2001

IL-1 beta- and IL-4-induced down-regulation of autotaxin mRNA and PC-1 in fibroblast-like synoviocytes of patients with rheumatoid arthritis (RA).

Autotaxin (ATX) is a 125-kD ectonucleotide pyrophosphate/phosphodiesterase, which was initially isolated and cloned from human melanoma cells as a potent stimulator of tumour cell motility. ATX shows 44% identity to the plasma cell membrane marker PC-1. Recently, we described the decreased expression of ATX mRNA in cultured fibroblast-like synoviocytes (SFC) of patients with RA by interferon-gamma. In this study using a competitive reverse transcriptase-polymerase chain reaction, we show an increased ATX mRNA expression in SFC from patients with RA in comparison with synoviocytes from non-RA patients. The median ATX mRNA amount in SFC of RA patients (440 pg/microg total RNA) was five-fold higher than the expression in synoviocytes from non-RA patients (80 pg/microg total RNA) or foreskin fibroblasts (MRHF cells, 90 pg/microg total RNA). In contrast to the elevated ATX mRNA expression in SFC of patients with RA, we did not measure increased mRNA amounts of PC-1 in these cells. Both the ATX mRNA amount and the 5'-nucleotide phosphodiesterase (PDE) activity of SFC lysate were reduced after treatment of SFC with the cytokines IL-1beta or IL-4. IL-1beta and IL-4 induced a down-regulation of PC-1 mRNA and protein expression in SFC. In SFC treated with transforming growth factor-beta the expression of PC-1 mRNA and protein was increased, whereas no significant effect on ATX mRNA expression was detectable. Pharmacological drugs used in therapy for RA, such as dexamethasone, cyclosporin, methotrexate and indomethacin, did not show a statistically significant effect on either ATX mRNA or PC-1 mRNA expression. Only pentoxifylline suppressed ATX mRNA as well as PC-1 mRNA expression. In conclusion, we show a tight regulation of ATX and PC-1 gene expression by cytokines detectable in the inflamed tissue of RA. Further investigations will deal with the regulation of ATX protein expression as well as with the function of ATX in RA.

Anti-Inflammatory Agents↗
PubMed1986

Patterns of region-specific chorion gene expression in the silkmoth and identification of shared 5' flanking genomic elements.

Time-dependent and cell-specific changes in concentrations of total RNA, of poly A+ RNA, and of specific mRNAs have been measured throughout silkmoth choriogenesis. Levels of total RNA and of poly A+ RNA are maintained throughout much of choriogenesis, but decrease at least fourfold during the very late period, in parallel with a decrease in overall protein synthesis. Very late period changes in total RNA and in poly A+ RNA are less pronounced in the aeropyle crown region, where a subset of chorion proteins is preferentially synthesized, than in the flat region. Maximal accumulation of the E1 and E2 chorion mRNAs occurs preferentially in the aeropyle crown region during the very late period. Uridine pulse-labeling studies suggest that E1 and E2 transcription is similarly aeropyle crown region-specific and immediately precedes the time of maximal E1 and E2 RNA accumulation and protein synthesis. Sequences from the 5' flanking regions of E1 and E2 genes have been compared. Several oligonucleotide sequences are present in both genes, and some are duplicated. These are potential cis-acting, regulatory elements.

Animals↗
PubMed2005

Colorimetric detection of eukaryotic gene expression with DNA-derivatized gold nanoparticles.

Thiol-linked DNA-gold nanoparticles were used in a novel colorimetric method to detect the presence of specific mRNA from a total RNA extract of yeast cells. The method allowed detection of expression of the FSY1 gene that encodes a specific fructose/H+ symporter in Saccharomyces bayanus PYCC 4565. FSY1 is strongly expressed when the yeast is grown in fructose as the sole carbon source, while cells cultivated in glucose as the sole carbon source repress gene expression. The presence of FSY1 mRNA is detected based on color change of a sample containing total RNA extracted from the organism and gold nanoparticles derivatized with a 15-mer of complementary single stranded DNA upon addition of NaCl. If FSY1 mRNA is present, the solution remains pink, changing to blue-purple in the absence of FSY1 mRNA. Direct detection of specific expression was possible from only 0.3 microg of unamplified total RNA without any further enhancement. This novel method is inexpensive, very easy to perform as no amplification or signal enhancement steps are necessary and takes less than 15 min to develop after total RNA extraction. No temperature control is necessary and color change can be easily detected visually.

Colorimetry↗
PubMed2006

The gene expression profile in refractory periodontitis patients.

BACKGROUND: There are no specific bacterial profiles or diagnostic tests capable of identifying refractory periodontitis patients before a treatment regimen is initiated. Therefore, in this high-risk cohort of patients who do not respond appropriately, host factors that might be partly under genetic control may play a crucial role in their susceptibility. Specifically, we tested the hypothesis that patients with refractory periodontitis have multiple upregulated and/or downregulated genes that might be important in influencing clinical risk. METHODS: Oral subepithelial connective tissues were harvested aseptically from seven refractory periodontitis and seven periodontally well-maintained patients. An RNA isolation kit was used to isolate total RNA from tissue samples that had been stabilized in the RNA stabilizing reagent. The isolated total RNA was then subjected to gene expression profiling using the microarray to measure gene expression levels. The retrieved data were analyzed with a computer program for the differential analysis of gene expression microarray experiments. In addition, real-time polymerase chain reaction (PCR) analysis was performed on selected samples to confirm the microarray data's gene expression patterns. RESULTS: A total of 68 upregulated and six downregulated genes were identified that were differentially expressed at least two-fold out of 22,283 genes we analyzed. The selected model provided a 93% intrinsic validation along with a 93% extrinsic validation. To validate the microarray data, five upregulated genes (lactotransferrin [LTF], matrix metalloproteinase-1 [MMP-1], MMP-3, interferon induced-15 [IFI-15], and Homo sapiens hypothetical protein MGC5566) and two downregulated genes (keratin 2A [KRT2A] and desmocollin-1 [DSC-1]) were randomly selected for further analysis by real-time PCR. The relative RNA expression level of these genes measured by real-time PCR was similar to those measured by microarrays. CONCLUSION: The combined use of microarray technology with the computer program for the differential analysis of gene expression microarray experiments provided a set of candidate genes that may serve as novel therapeutic intervention points and improved diagnostic and screening procedures for high-risk individuals.

Aged↗
PubMed2004

Antitumor effects of vaccine consisting of dendritic cells pulsed with tumor RNA from gastric cancer.

AIM: To investigate the immunotherapeutic potential of vaccine consisting of dendritic cells (DCs) pulsed with total RNA from MFC gastric cancer cells. METHODS: DCs were prepared from the spleens of strain 615 mice by magnetic cell sorting (MACS). After culture for 24 h, DCs were pulsed with total RNA from MFC gastric cancer cells. Mice of one group were immunized with tumor RNA pulsed DC (RNA/DC) at the dosage of 1X10(6) on d 14 and 7 by s c inoculation before tumor implantation. Mice of another group were immunized with unpulsed DC (UDC) at the same dosage on days as the RNA/DC group. The third group of control mice was untreated. On d 0, all the mice were challenged with s c injections of 5X10(5) MFC gastric cancer cells. After inoculation, the mice were monitored closely with respect to tumor growth. Activities of NK cells in PBL and splenocytes and CTL were tested. RESULTS: On d 21 after tumor cell inoculation, the mice of control group manifested the largest tumors with volume at a mean of 2.6323+/-1.1435 cm(3), followed by the UDC and RNA/DC groups with mean volumes at 0.7536+/-0.3659 cm(3) and 0.3688+/-0.6571 cm(3), respectively. The activities of NK cells in PBL and splenocytes in RNA/DC group were 66.2% and 65.4%, respectively, higher than that in the control group. The tumor specific CTL activity in RNA/DC group was 49.5%, higher than that in the control group. CONCLUSION: The tumor vaccine with DCs pulsed with total RNA from gastric cancer cells possesses the ability to stimulate tumor specific CTL activity and to establish anti-tumor immunity when administered in vivo.

Animals↗
PubMed1998

Regional expression of disease-related genes in human and monkey retina.

PURPOSE: Although specific genes play a role in regional retinal disease, the correlation of regional gene expression in the disease-affected site has not been previously ascertained. Non-human primates are widely used in models of human retinal function and are theorized to have identical (to human) patterns of expression, but no correlation between primate and human regional retinal gene expression has ever been performed. We wanted to evaluate the pattern of regional gene expression for a number of genes whose dysfunctions are known to selectively affect specific regions of the human retina, and to determine whether patterns of regional gene expression in nonhuman primates correlate with the human. METHODS: Human and rhesus monkey eyes were dissected into retina, retinal pigment epithelium (RPE)/choroid and isolated RPE. Retinal regions were dissected, total RNA was isolated and northern analysis performed. Complementary DNA (cDNA) probes were prepared from genes associated with regional retinal disease. These genes are: rod opsin, the alpha-subunit of rod phosphodiesterase, RDS-peripherin, rod outer membrane (ROM) protein, ornithine aminotransferase (OAT), choroideremia gene product (CHM), tissue specific inhibitor of metalloproteinases-3 (TIMP-3), and red/green photoreceptor pigment protein. We also compared expression of Norrie disease product (NDP), a gene whose mutation is known to globally affect the retina. RESULTS: Rod-specific mRNA expression is highest in the retinal midperiphery, and cone-specific mRNA levels were highest in total RNA from the cone-dominant fovea. mRNA levels for genes coding for proteins expressed in both rod- and cone photoreceptors (RDS-peripherin and ROM-1) are also highest in total RNA from the retinal midperiphery. Regional mRNA levels of CHM and OAT do not directly correlate with their patterns of disease expression. NDP mRNA expression was equivalent in both fovea and midperipheral retina total RNA. Patterns of gene expression were qualitatively similar for both human and rhesus monkey retina. CONCLUSIONS: Regional retinal gene expression is an important factor in regional disease. However, for genes not solely expressed by a single photoreceptor subtype, other factors, such as regional metabolic differences, intra- and intercellular interactions, are also likely to be important in predisposing a single retinal region to disease. The pattern of neural retina OAT mRNA expression may have important implications in determining the appropriate tissue approach in gene therapy for gyrate atrophy. Regional retinal gene expression likely plays a significant, but nonexclusive role in the development of regional retinal disease.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Some properties of RNA from Physarum amoebae undergoing encystment: electrophoretic similarity to Acanthamoeba RNA.

Some properties of the RNA of myxamoebae-swarm cells of Physarum flavicomum undergoing microcyst formation, and during adenine inhibition of the developmental process, were compared. During the first 15 h incubation under encystment conditions, 11% more RNA was recovered from the adenine-inhibited cells (AIC) than from the normal control cells (NC), whereas the specific activity (SA) of the purified RNA from NC was 1.4 times higher than that from the AIC. Gel electrophoresis of purified total RNA revealed comigration of ribosomal RNAs (rRNA) of the myxamoebae with those of mouse kidney and liver, and diploid cells of P. flavicomum. In contrast to the apparently equivalent 18S and 28S pattern of the latter three RNAs, the quantity of the largest rRNA was significantly lower compared with that of the smaller (18S) rRNA in both NC and AIC myxamoebae-swarm cells. Total RNA from amoebae of a recent isolate of Acanthamoeba sp. quantitatively exhibited the same unusual pattern of rRNA distribution as the Physarum myxamoebae. A high pressure liquid chromatographic method was developed using a single solvent for the isocratic separation of the four major bases and seven methylated bases of RNA. Methylated adenine and guanine were detected in total RNA samples of myxamoebae of P. flavicomum. The mole % of uracil was slightly higher in the NC, whereas the mole % of adenine was slightly lower compared with that of the AIC. The guanine plus cytosine content of these purified total RNAs was about 61%.

Acanthamoeba↗
PubMed1988

Transcriptional activation of the glutathione S-transferase pi gene in human ureteric and bladder carcinomas.

Using a cDNA clone derived from the rat Glutathione S-Transferase P (GST-P) gene, we have investigated homologous transcripts in a range of normal and malignant human tissues. A major transcript, of 0.75 Kb, representing human GST-pi mRNA, was present in total RNA purified from peripheral white blood cells, bladder, ureteric and prostate tissues. In total RNA derived from histopathologically benign/normal tissues, peripheral leucocytes and from hyperplastic and malignant prostates, levels of the mature transcript varied by less than 1.5-fold (n = 70). In contrast, levels of transcript were significantly elevated (up to 16-fold) in total RNA derived from bladder and ureteric carcinomas, with the highest levels of elevation approaching those previously found only for the GST-P gene in experimentally induced rodent hepatocellular carcinomas. A wide range of stage and grade of tumour has been investigated (pT1 to pT4; G1 to G3, respectively), suggesting that the transcriptional activation of the GST-pi gene is a characteristic common to many bladder/ureteric carcinomas in man.

Carcinoma↗
PubMed2004

Dexamethasone regulation of gastrin-releasing peptide receptor in human lung cells.

We investigated the effects of the glucocorticoid, dexamethasone (Dex), on expression of the gastrin-releasing peptide (GRP) receptor by human small cell lung carcinoma (SCLC) SHP77 cells. After 12h of 10nM Dex exposure, a six-fold increase in the peak of GRP receptor mRNA compared with untreated controls (10.5+/-4 versus 1.65+/-0.15 attomols/microg total RNA, respectively, P<0.05) occurred. GRP receptor mRNA levels fell to less than 0.5 attomols/microg total RNA after 24h; in Dex-treated cells, these levels rose to 1.2 compared with 0.12 attomols/microg total RNA in the absence of Dex after 7 days. A significant increase (P<0.05) in the GRP receptor-specific binding was also found. Stimulation of SHP77 cell proliferation (25-35% in the presence of 10-100 nM Dex; P<0.0001) was observed after 4-8 days of exposure; this stimulation was inhibited by GRP receptor antagonists. SHP77 cell content and concentration of bombesin-like peptides (BLP) in conditioned medium (approximately 4 nM) was unchanged by Dex. Stimulation of human SCLC SHP77 cell proliferation by Dex may, in part, occur via effects on the GRP autocrine system in these cells.

Cell Division↗
PubMed1999

Attenuated expression of 70-kDa heat shock protein in WI-38 human fibroblasts during aging in vitro.

We examined the effects of cellular aging on the expression of the heat shock-inducible HSP70 gene in WI-38 diploid human fibroblasts serially passaged in vitro. The senescence of the cells was established by evaluating population doubling level, cell density at confluency, and cell morphology along with the detection of senescence-associated beta-galactosidase activity (histochemically detectable at pH 6), a reliable marker of aging in low-density cultures. A marked decrease in the synthesis and accumulation of the inducible HSP70 protein was observed in serum-fed late passage cells exposed to a severe heat shock (30 min at 45 degrees C) in comparison to early passage cells. However, the degree of HSF-DNA binding, monitored by gel retardation assay was similar in both early and late passage cells. Similarly, Northern blotting analysis indicated that comparable amounts of inducible HSP70 mRNA were present in the total RNA fraction, in the total polyadenylated RNA fraction, or in the nuclear polyadenylated RNA fraction extracted from both early and late passage cells. In contrast, much less inducible HSP70 mRNA was detected in the total cytoplasmic RNA fraction or in the polyadenylated cytoplasmic RNA fraction of late passage cells. Thus age-related differences in heat-induced HSP70 synthesis and accumulation observed in serum-fed WI-38 cells appeared to result from an impairment in the posttranscriptional processing of the HSP70 mRNA at a level following the polyadenylation step and preceding translocation from the nucleus to the cytoplasm. When HF were serum deprived for 20 h before heat shock, the induction of HSP70 mRNA was less than 30% reduced in early passage cells in comparison to serum-fed cells; however, the level of HSP70 mRNA was markedly (over 80%) decreased in serum-deprived late passage cells. This result indicated that the presence of serum has a strong influence on heat shock-induced HSP70 gene expression in human fibroblasts aging in vitro.

Base Sequence↗
PubMed1997

Modified Method for Competitive Reverse Transcription Polymerase Chain Reaction for Rapid and Automated Quantitation of Messenger RNA in Multiple Samples.

Background: Competitive reverse transcription polymerase chain reaction (RT-PCR) has been used increasingly to quantitate messenger RNA (mRNA) levels; however, conventional competitive RT-PCR methods require four or five reactions per sample of RNA, employing serial dilutions of an internal competitor sequence, making analysis of multiple samples of tedious process. A modified method is described by which multiple samples and multiple RNA transcripts can be analyzed easily by an automated process. Methods and Results: Transforming growth factor beta-1 (TGF-beta-1) mRNA was assayed in total RNA extracted from cultured human skin fibroblasts. A standard solution of total RNA was first prepared by pooling RNA from several cell lines and stored in aliquots. A 270-bp competitor RNA molecule (RNA mimic) was prepared by in vitro transcription and was added to each reaction. PCR was performed with a fluorescent dye (Hex; Applied Biosystems, Foster City, CA)-labeled sense primer to amplify a 161-bp-long c DNA product of target TGF-beta-1 mRNA sequence and the RNA mimic. The PCR products were analyzed with an automated laser-scanned gel electrophoresis system and the area under the curve (AUC) was used for quantitation. The concentration TGF-beta-1 mRNA in standard RNA was determined by conventional competitive RT-PCR. Subsequently, equal amounts of RNA mimic were mixed with four serial dilutions of standard RNA and 0.1 µg of sample total RNA for RT-PCR. A standard curve was generated using the known dilutions of a standard target RNA solution and ratio of AUC for target to that for mimic for each dilution. The unknown sample was then quantitated by interpolation of its area under the curve ratio on the standard curve. This method had inter- and intra-assay coefficients of variation of less than 10%. Conclusions: This modification is highly reproducible for quantitation of mRNA and significantly reduces the number of PCR reactions required for each assay. It can be used to assay several RNA molecules in a given sample by designing RNA mimics and PCR primers to generate PCR products of different lengths so that they can be analyzed by the laser scanning of a single lane of electrophoretic gel.

Journal Article↗
PubMed2000

Regulation of uncoupling protein-2 and uncoupling protein-3 mRNA expression during lipid infusion in human skeletal muscle and subcutaneous adipose tissue.

To study the effect of nonesterified fatty acids (NEFAs) on uncoupling protein-2 (UCP-2) and uncoupling protein-3 (UCP-3) gene expression, a triglyceride emulsion was infused for 5 h in 14 healthy volunteers. A euglycemic-hyperinsulinemic clamp was administered concomitantly in 7 of the 14 subjects. The mRNA levels of UCP-2 and of the short (UCP-3S) and long (UCP-3L) isoforms of UCP-3 were quantified by reverse transcription-competitive polymerase chain reaction in tissue biopsies taken before and at the end of the infusion periods. Plasma NEFA concentrations increased from 362+/-52 to 989+/-157 micromol/l (P = 0.018) during triglyceride infusion. UCP-3L (8+/-1 vs. 19+/-2 amol/microg total RNA, P = 0.018) and UCP-3S (11+/-2 vs. 17+/-3 amol/microg total RNA, P = 0.027) mRNA levels increased in skeletal muscle during triglyceride infusion. UCP-3L mRNA levels were positively correlated with plasma NEFA concentrations (r = 0.53, P = 0.005) and with lipid oxidation rates (r = 0.56, P = 0.004) determined by indirect calorimetry. In contrast, the expression of UCP-2 was not affected by lipid infusion in skeletal muscle or in subcutaneous adipose tissue. During the hyperinsulinemic clamp (plasma insulin concentrations 202+/-12 pmol/l), NEFA levels (405+/-49 vs. 648+/-77 micromol/l, P = 0.063) and lipid oxidation rates (0.67+/-0.09 vs. 0.84+/-0.10 mg x kg(-1) x min(-1), P = 0.091) were not significantly increased during triglyceride infusion. Under such conditions, the induction of UCP-3L and UCP-3S mRNA expression was totally prevented (8+/-2 vs. 8+/-1 and 8 +/-2 vs. 9+/-2 amol/microg total RNA, respectively). We conclude that increased plasma NEFA levels by lipid infusion for 5 h induces the expression of UCP-3 but not UCP-2 in humans. During triglyceride infusion, physiological hyperinsulinemia appears to prevent the induction of UCP-3 mRNA levels.

Adipose Tissue↗
PubMed2006

Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses.

Recent research indicates that dendritic cells transfected with RNA-encoded tumour-associated antigens (TAA) can generate potent anti-tumour immune responses in vitro and in vivo. RNA is an important source of TAA, but its relatively unstable nature, in addition to often limited availability of tumour tissue, may represent a considerable obstacle for its use. Our first goal was to establish an efficient protocol for the preparation of high quality total RNA from tumour samples. This should then be used as such or be pre-amplified for DC transfection. Therefore native total RNA was prepared from stabilised tissue samples obtained from liver metastases of colon cancer using either solution- or silicagel-based protocols for RNA isolation. The first isolation protocol yielded higher amounts of total RNA, but with lower purity as compared to the second one. No degradation of RNA was observed regardless of the protocol used. Subsequently, we focused on the amplification of mRNA. The fidelity of the amplified mRNA was confirmed by RT-PCR for glyceraldehyde-3-phosphate-dehydrogenase (GADPH) and carcinoembryonic antigen (CEA) coding sequences. We found no differences in the induction of CEA-specific CTL responses between native and amplified RNA-transfected DCs. Additionally, we tested the induction of CTL responses and found that DCs transfected with amplified mRNA originating from either tumour tissue or a cell line were able to induce strong anti-tumour CTL responses in vitro. They were comparable to those induced by native total RNA-transfected DCs. Our results therefore indicate that the amplified mRNA is equivalent to the native one in the induction of anti-tumour CTL responses and can be used for generation of RNA-transfected DCs.

Cell Line, Tumor↗
PubMed2006

RNA integrity and the effect on the real-time qRT-PCR performance.

The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application.

Animals↗
PubMed2005

Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications.

BACKGROUND: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3'-Ribo-SPIA primes cDNA synthesis at the 3' polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3' polyA tail. METHODS: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3'-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA). RESULTS: Ribo-SPIA-based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA. CONCLUSIONS: The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non-polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.

Gene Expression Profiling↗
PubMed2003

Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells.

In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from naïve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.

Antigen-Presenting Cells↗
PubMed1982

Effects of histone hyperacetylation and hypoacetylation on RNA synthesis in HTC cells.

We have analyzed the RNA synthetic activity in intact HTC cells which have artificially high or low levels of histone acetylation. Rates of total RNA synthesis and the number of nascent transcription complexes have been measured for 20-h butyrate-treated cells and for butyrate-released cells which contain hypoacetylated histones. Exposure of HTC cells to butyrate for 20 h leads to a approximately 40% decrease in the number of nascent RNA molecules and a corresponding approximately 40% decrease in the rate of total RNA synthesis. During the induction of hypoacetylation following the release of cells from butyrate, rates of RNA synthesis and the number of nascent transcripts remain unchanged. Between the fifth and eighth h of release from butyrate, the number of nascent transcripts and the rate of total RNA synthesis increase to control cell levels. This increase in the number of nascent RNA polymerase complexes, while occurring when bulk histone is hypoacetylated, is temporally correlated with an increase in the rate of acetylation for a subset of histones. In contrast, changes in levels and rates of histone acetylation were found to be without effect on the average rate of RNA chain elongation. Additionally, we have found that the complexity of nuclear RNA is unchanged by a 20-h exposure of HTC cells to butyrate, suggesting that the approximately 40% decrease in frequency of nascent polymerase complexes produced by butyrate does not result from the complete loss of transcription of a large number of genes.

Acetylation↗
PubMed2001

[Construction of recombinant antisense RNA IgG2aVH region of murine systemic lupus erythematous].

OBJECTIVE: To construct recombinant plasmid of IgG2aVH region antisense RNA of systemic lupus erythematous. METHODS: Total RNA was isolated from spleen cell of BWF1 mice. Using spleen cell total RNA as the template, We designed specific primers from A6.1 region sequences, and amplified the IgG2aVH region 375 bp DNA fragments by RT-PCR. The IgG2aVH cDNA was cloned by T/A and inserted into pcDNA 3.1 plasmid of vector. RESULTS: The IgG2aVH antisense RNA plasmid expressing recombinant was identified by restriction enzyme digestion and DNA sequence analysis. CONCLUSION: There is IgG2aVH gene in F1 mice spleen cell; the IgG2aVH antisense RNA expressing recombinants is constructed successfully.

Animals↗