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PubMed2001

Prolactin receptor gene expression and immunolocalization of the prolactin receptor in human luteinized granulosa cells.

Prolactin is mainly known for its role in breast development and lactation, but has been also implicated in other physiological functions such as immunoregulation and ovarian steroid production. Although prolactin and prolactin receptor (PRL-R) transcripts have been previously identified in the human ovary, the spatial localization of the receptor is unknown. To investigate the presence of PRL-R within the follicular apparatus, human luteinized granulosa cells were obtained at the time of follicular aspiration from women undergoing ovarian stimulation for IVF. RNA extracted from these cells was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the PRL-R gene. In addition, paraffin sections of isolated granulosa cells and sections of premenopausal human ovaries were immunostained with a mouse anti-human PRL-R monoclonal antibody. PRL-R were immunolocalized to the cell membrane of isolated luteinized granulosa cells and PRL-R transcripts were detected in the extracted RNA. No detectable staining was noted in secondary and early antral follicles in archived paraffin sections. These findings confirm the presence of PRL-R in human luteinized granulosa cells and suggest a localized role for PRL within the mature follicle. The absence of PRL-R in the early follicle suggests that the effects of prolactin are exerted around the time of ovulation.

Adult↗
PubMed1997

Posttransplant diastolic hypertension: associations with intragraft transforming growth factor-beta, endothelin, and renin transcription.

BACKGROUND: Diastolic hypertension after renal transplantation leads to significant chronic morbidity and mortality. Recently, calcineurin phosphatase inhibition by cyclosporine or tacrolimus has been postulated to lead to diastolic hypertension through the induction of transforming growth factor-beta (TGF-beta) and resultant endothelin-mediated renal arteriolar vasospasm. METHODS: To investigate this hypothesis in humans, the allografts of 40 stable renal allograft recipients were biopsied 2 to 3 years after transplantation. Both cyclosporine and tacrolimus patients were included. Biopsies were divided and processed for histology and RNA extraction. RNA was then converted to cDNA and evaluated by semiquantitative polymerase chain reaction (actin-standardized, high-performance liquid chromatography-quantitated) for TGF-beta, endothelin, and renin transcription. Inflammatory cytokine gene transcription was also evaluated. Blood pressure was measured during the clinic check-in before biopsy. Variables were evaluated by Spearman rank correlation coefficient (rs) analysis. RESULTS: Diastolic hypertension was prevalent in the study population, with 40% of individuals having diastolic pressure >90 mmHg. TGF-beta and endothelin transcription were detected in 88% of biopsies studied, and renin transcription was detected in 91%. Intragraft transcription of TGF-beta (rs=0.61, P=0.0003) and endothelin (rs=0.43, P=0.0188) was strongly correlated with increasing transcription intragraft renin. In turn, renin transcription was strongly correlated with increasing diastolic blood pressure (rs=0.55, P=0.0015). Histological correlation of fibrosis score did not predict the degree of hypertension, nor did it correlate with TGF-beta transcription. Inflammatory cytokine transcription was not related to renin transcription or diastolic hypertension but was correlated with histological evidence of immune graft injury. CONCLUSIONS: These data support the hypothesis that posttransplant diastolic hypertension is a result of TGF-beta-induced, endothelin-mediated arteriolar vasoconstriction and subsequent activation of the renin-angiotensin pathway. These effects are independent of immune-mediated graft injury.

Adult↗
PubMed1999

Clinically stable human renal allografts contain histological and RNA-based findings that correlate with deteriorating graft function.

BACKGROUND: Chronic rejection (CR) remains idiopathic, difficult to prospectively identify, and once detected, unresponsive to increased immunosuppression. We hypothesized that clinically stable human renal allografts have ongoing evidence of injury and immune activity, and that this correlates with the worsening of allograft function characteristic of CR. METHODS: The allografts of 40 stable renal allograft recipients were biopsied 2-3 years after transplantation. Biopsies were processed for histology and RNA extraction. RNA was evaluated by semi-quantitative RT-polymerase chain reaction for CD3y mRNA (a marker of T cell receptor turnover), and mRNA from cytokine genes previously shown to be transcribed during acute rejection: tumor necrosis factor-alpha, interferon-gamma, interleukin- (IL) 1beta, IL-2, IL-4, IL-6, and IL-8. Clinical parameters including urine protein and glomerular filtration rate were measured the day of biopsy. Findings were then compared with clinical outcome to establish associations between subclinical inflammation and graft dysfunction. Allograft function was measured again 2 years after biopsy and correlated with findings at the time of biopsy. RESULTS: Cytokine transcripts and histological evidence of injury were detected in more than two-thirds of stable grafts. The degree of the lymphocytic infiltrate correlated with the degree of proteinuria (P=0.034) and histological fibrosis (P=0.005). Similarly, the degree of intragraft CD3y transcription correlated with increasing proteinuria (P=0.043). IL-6 and IL-8 transcripts were also correlated with evidence of graft injury. After 2 years, those biopsies originally found to have evidence of fibrosis, tubular atrophy, or CD3gamma transcription had worsening graft function as determined by creatinine and glomerular filtration rate. CONCLUSIONS: These data demonstrate that significant injury and immune activity can be detected in patients who are stable on clinical grounds. Undetected subclinical graft injury may be a cause of chronic allograft rejection.

Biomarkers↗
PubMed1994

Expression of the gene for inducible nitric oxide synthase in experimental glomerulonephritis in the rat.

Nitrite, a stable product of nitric oxide (NO), is synthesized in vitro by glomeruli in experimental glomerulonephritis. We have now studied the expression of the gene for inducible NO synthase (iNOS) in accelerated nephrotoxic nephritis (NTN). The purpose of the study was to confirm in vivo induction of iNOS in this model of immune complex disease, and to relate the onset of induction and the level of expression to pathogenic events in the model. Glomeruli from rats with NTN were isolated at 6 h, 24 h and 2, 4 and 7 days and total RNA extracted. RNA (10 micrograms) was reverse transcribed and polymerase chain reaction (PCR) was performed with primers homologous to rat vascular smooth muscle iNOS and rat beta actin. A 222-base PCR product corresponding to iNOS mRNA was present in all experimental animals. iNOS expression was also found in activated macrophages, neutrophils and IL-1-stimulated but not unstimulated mesangial cells. Quantitative competitive PCR was carried out on glomerular samples using a 514-bp mutant of a 735-bp PCR product. iNOS expression was present at low levels in normal glomeruli and was markedly enhanced at 6 h after the induction of glomerulonephritis and peaked at 24 h. Increased iNOS expression persisted to day 7. beta actin mRNA levels were similar in all glomerular specimens. This study demonstrates that there is in vivo induction of iNOS in immune complex glomerulonephritis, corresponding to the generation of nitrite we have previously reported. iNOS gene expression is detectable within 6 h of induction of NTN, indicating the onset of gene transcription is closely related to the initial formation of immune complexes.

Amino Acid Oxidoreductases↗
PubMed2004

A novel viral transcript with homology to human interleukin-10 is expressed during latent human cytomegalovirus infection.

Human cytomegalovirus (CMV) establishes latent infections in hematopoietic cells such as granulocyte-macrophage progenitors (GM-Ps). During latency the virus is sequestered in a nonreplicating state, although limited transcriptional activity has been previously reported. In this study we sought to further examine viral gene expression during the latent phase of infection. Using an experimental model of latency, primary human GM-Ps were latently infected with CMV strain Toledo and extracted RNA subjected to reverse transcription-PCR by using CMV gene-specific primers. Using this approach, we detected transcription from the UL111.5A region of the viral genome. This transcription was also detected in GM-Ps latently infected with AD169 and Towne strains, indicating that expression was CMV strain independent. Significantly, we detected UL111.5A-region transcripts in mononuclear cells from healthy bone marrow and mobilized peripheral blood allograft donors, demonstrating expression during natural latent infection. Mapping experiments with RNA extracted from latently infected GM-Ps revealed the expression of a novel UL111.5A region transcript with a splicing pattern that differed from that reported during productive infection of permissive cells. This UL111.5A region transcript expressed during latent infection is predicted to encode a 139-amino-acid protein with homology to the potent immunosuppressor interleukin-10 (IL-10) and to the viral IL-10 homolog that is expressed during productive CMV infection. Expression of a latency-associated cmvIL-10 may confer upon the virus an ability to avoid immune recognition and clearance during the latent phase of infection.

Amino Acid Sequence↗
PubMed2004

Partial sciatic nerve transection.

Partial sciatic nerve transection (PST) of the sciatic nerve of rats and mice is described as a model of painful neuropathy. The rationale for developing this model was to establish a simple partial nerve injury without application of foreign material. In contrast to the frequently used model of chronic constriction injury (CCI), PST allows to relate animal behavior and drug effects to endoneurial changes, undisturbed by major epineurial inflammation. PST is easy to perform in rats and mice and leads to reproducible pain-related behavior for 5 wk or longer.

Animals↗
PubMed2004

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR.

Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

Animals↗
PubMed2004

[A HLA-A2-restricted-CTL epitope variation at the N-terminal of HCV helicase and the immune response of CTLs].

AIM: To investigate a HLA-A2-restricted CTL epitope variation at the N terminal of hepatitis C virus (HCV) helicase and the proliferative response of cytotoxic T lymphocytes (CTLs) to this epitope. METHODS: Two patients infected with HCV were followed up for 7 years. Blood samples taken at the first year and the fifth year were used for viral RNA extraction. Using RT-PCR, the N terminal gene of helicase region was amplified from the extracted RNA. The amplified segments were subcloned and sequenced to assess the epitope variation. A HLA-A2-restricted CTL epitope peptide was synthesized according to the sequencing result. HLA types of the two patients were determined by serological method. The peripheral blood lymphocytes were separated from the two patients at 7th year after infection and cultured with the synthetic CTL epitope peptide to assess the CTL's proliferative response. RESULTS: In the HLA-A2(+) individual, there was an amber mutation at the first amino acid of the epitope in the first year after infection, but the amber mutation disappeared after 5 years. However, in the HLA-A2(-) individual, the epitope had neither variation nor amber mutation in 5 years after infection. 7 years after HCV infection, T lymphocytes from both individuals had no proliferative response to the epitope peptide. CONCLUSION: The amber mutation of the epitope at N terminal of helicase may be associated with viral immune escape. CTLs have no response to the epitope peptide in the N terminal of HCV helicase in the later stage of chronic HCV infection.

Adult↗
PubMed1974

Formation of Sindbis virus capsid protein in mammalian cell-free extracts programmed with viral messenger RNA.

Extracts from Krebs II ascites cells and rabbit reticulocytes effectively synthesize viral proteins with Sindbis viral mRNA isolated from Sindbis-infected BHK cells. The major product is identical to Sindbis capsid protein on the basis of its electrophoretic mobility in sodium dodecyl sulfate-acrylamide gels and two-dimensional tryptic-peptide fingerprints. Various amounts of several additional discrete polypeptides are formed, depending on the components of the cell-free extracts. One of these polypeptides may be a prematurely terminated part of the viral-capsid protein, while another is larger in molecular weight than capsid protein but contains the capsid tryptic peptides. Several of the proteins formed in vitro also are detected in extracts of Sindbis-infected BHK cells labeled with [(35)S]methionine. The three proteins found in Sindbis virions are postulated to originate by proteolytic cleavage from a larger molecular weight polypeptide precursor that is translated from a polycistronic mRNA presumed to contain a single site for initiation of protein synthesis. The two in vitro systems appear to translate this polycistronic viral mRNA to yield specific viral capsid although no evidence was found for post-translational proteolysis. Other mechanisms for production of the capsid protein in the cell-free extracts are considered, and some of these may function in the viral-infected cell where unusually large amounts of viral capsid proteins are frequently detected.

Animals↗
PubMed2025

Systematic comparison of quantity and quality of RNA recovered with commercial FFPE tissue extraction kits.

BACKGROUND: FFPE tissue samples are commonly used in biomedical research and are a valuable source for next-generation sequencing in oncology, however, extracting RNA from these samples can be difficult the quantity and quality achieved can impact the downstream analysis. This study compared the effectiveness of seven different commercially available RNA extraction kits specifically designed for use with FFPE samples in terms of the quantity and quality of RNA recovered. METHODS: This study used 9 samples of FFPE tissue from three different types of tissue (Tonsil, Appendix and lymph node of B-cell lymphoma) to evaluate RNA extraction methods. Three sections of 20 µm of each sample were combined per sample. The slices were distributed in a systematic manner to prevent any biases. Each of the 7 commercially available RNA extraction kits were used according to manufacturer's instructions, with each sample being tested in triplicate resulting in a total of 189 extractions. The concentration, RNA quality score (RQS) and DV200 of each extraction was analysed using a nucleic acid analyser to determine the quantity and quality of the recovered RNA. RESULTS: This study found that despite processing the FFPE samples in the same standardized way, there were disparities in the quantity and quality of RNA recovered across the different tissue types. Additionally, the study found notable differences in the quantity of RNA recovered when using different extraction kits. In terms of quality, three of the kits performed better than the other four in terms of RQS and DV200 values. CONCLUSION: Though many laboratories have developed their own protocols for specific tissue types, using commercially available kits is still a popular option. Although these kits use similar processes and extraction procedures, the amount and quality of RNA obtained can vary greatly between kits. In this study, among the kits tested, while the Roche kit, provided a nearly systematic better-quality recovery than other kits, the ReliaPrep FFPE Total RNA miniprep from Promega yielded the best ratio of both quantity and quality on the tested tissue samples.

Paraffin Embedding↗
PubMed1986

Transcription of genomic bovine and Xenopus laevis DNA species by RNA polymerase III in HeLa-cell cytosol extracts.

RNA transcribed from genomic Xenopus laevis DNA by RNA polymerase III in HeLa-cell extract was found in discrete size classes and was transcribed from at least two different Xenopus repeat DNA species. Very little 5S ribosomal RNA was transcribed, contrasting with results obtained on transcription of genomic Xenopus DNA by Xenopus extract [Bogenhagen et al. (1982) Cell 28, 413-421]. The low transcription was not due to an inability to use 5S rDNA templates, since the cloned Xenopus 5S ribosomal gene and pseudogene were effective templates for RNA polymerase III in HeLa extract. RNA transcribed from genomic bovine DNA by RNA polymerase III in HeLa-cell cytosol extract consisted of 120-nucleotide RNA and a larger amount of heterogeneously sized RNA (180-650 nucleotides). Only a small portion of the 120-nucleotide RNA was 5S rRNA. Most of the 120-nucleotide RNA and the larger RNA species were transcribed from one bovine repeat DNA. Genes for 5S rRNA and bovine repeat DNA were transcribed roughly in proportion to their frequency in Bos, contrasting with results in a homologous system in which transcription of repeat genes is repressed [Furth (1985) Biochem. Biophys. Res. Commun. 131, 551-556]. Bovine 5S rRNA genes appear to be concentrated on one DNA fragment obtained by restriction-enzyme-HindIII digestion of genomic bovine DNA.

Animals↗
PubMed2002

Rapid isolation of cancer cells from tumor tissue by micromanuiplator and extraction of tiny amount of RNA.

OBJECTIVE: To establish a rapid method for isolating and purifying cancer cells from tumor tissue and for RNA extraction from tiny amount of the cells thus obtained. METHODS: Frozen sections of the tumor tissues were prepared followed by rapid staining. Clusters of the cancer cells were isolated from the sections by micromanipulation technique and purified for extracting intact RNA that was subsequently assayed. RESULTS: Clear vision was achieved by the staining of the sections. The cancer cell clusters were precisely isolated from which high-quality intact RNA was obtained as demonstrated by reverse transcriptase-PCR. CONCLUSION: Micromanuiplation can be effectively used in stead of laser capture microdissection to isolate and purify targeted cells from tiny amount of tissue samples, therefore making RNA extraction possible in this context.

Electrophoresis, Agar Gel↗
PubMed1995

Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

Animals↗
PubMed1999

Major interference from leukocytes in reverse transcription-PCR identified as neurotoxin ribonuclease from eosinophils: detection of residual chronic myelogenous leukemia from cell lysates by use of an eosinophil-depleted cell preparation.

BACKGROUND: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 10(5) leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes. METHODS: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque(R) density gradient. RESULTS: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by approximately 90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 10(5) CD16(+) granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control. CONCLUSION: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.

Amino Acid Sequence↗
PubMed2003

A two-step method for the extraction of high-quality RNA from endoscopic biopsies.

The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a column-based extraction method, resulted in a yield of 4.5 microg total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR analysis revealed that only RNA samples that clearly show both 18S- and 28S-RNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug resistance protein-1 (mdr-1)-mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

A method to detect low levels of enteric viruses in contaminated oysters.

Direct isolation and identification of pathogenic viruses from oysters implicated in gastroenteritis outbreaks are hampered by inefficient methods for recovering viruses, naturally occurring PCR inhibitors, and low levels of virus contamination. In this study we focused on developing rapid and efficient oyster-processing procedures that can be used for sensitive PCR detection of viruses in raw oysters. Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy of virus recovery and the removal of PCR inhibitors during oyster-processing procedures. These procedures included elution, polyethylene glycol precipitation, solvent extraction, and RNA extraction. Acid adsorption-elution in which glycine buffer (pH 7.5) was used was found to retain fewer inhibitors than direct elution in which glycine buffer (pH 9.5) was used. RNA extraction in which a silica gel membrane was used was more effective than single-step RNA precipitation for removing additional nonspecific PCR inhibitors. The final 10-microl volume of RNA concentrates obtained from 2 g of oyster tissue (concentration factor, 200-fold) was satisfactory for efficient reverse transcription-PCR detection of virus. The overall detection sensitivity of our method was 1 PFU/g of oyster tissue initially seeded with PV3. The method was utilized to investigate a 1998 gastroenteritis outbreak in California in which contaminated oysters were the suspected disease transmission vehicle. A genogroup II Norwalk-like virus was found in two of three recalled oyster samples linked by tags to the harvest dates and areas associated with the majority of cases. The method described here improves the response to outbreaks and can be used for rapid and sensitive detection of viral agents in outbreak-implicated oysters.

Animals↗
PubMed1981

Antigenic modification, rosette-forming cells, and Salmonella typhimurium resistance in outbred and inbred mice.

To assess the separate contributions of host T cells and the physical state of the antigen in the development of effective. Salmonella resistance, glutaraldehyde-treated and untreated protein- and ribonucleic acid-rich extracts (E-RNA extracts) of virulent Salmonella typhimurium SR-11 or attenuated S. typhimurium RIA were used to immunize Salmonella-resistant Salmonella-susceptible strains of mice for the purpose of determining whether antigen-specific T-cell or B-cell responses were formed and, if so, which responses predominated. The resistance imparted to each mouse strain after vaccination with S. typhimurium RIA was used as the standard for comparison. The inbred mouse strains C57BL/6 and DBA/2 and their F(1) hybrid (strain BDF(1)), outbred ICR Swiss mice, and endotoxin-resistant C3H/HeJ mice were examined for the capacity to develop resistance to lethal Salmonella infections, as well as the ability to generate antigen-reactive T cells. Only the BDF(1), C3H/HeJ, and ICR Swiss mice were able to develop resistance to challenge infections mediated by the virulent SR-11 strain of S. typhimurium after vaccination with the living, attenuated RIA strain of S. typhimurium or immunization with E-RNA extracts. We developed an assay to identify the antigen-reactive rosette-forming lymphocytes present in lymph nodes and spleens of immunized mice. Levels of 0.2% or higher of theta antigen-bearing, antigen-reactive rosette-forming cells were found in the lymph nodes or spleens or both of only the BDF(1), C3H/HeJ, and ICR Swiss mice (i.e., in the "Salmonella responder" strains). Mouse strains C57BL/6 and DBA/2, which failed to develop resistance to lethal infections after immunization with the S. typhimurium RIA vaccine or with the E-RNA extracts, lacked effective numbers of antitheta antigen-sensitive rosette-forming cells. Modification of the effective E-RNA extracts by polymerization with glutaraldehyde resulted in a marked diminution in their abilities to induce resistance to salmonellosis in the two responder mouse strains tested (BDF(1) and ICR Swiss), even though detectable levels of antibody were induced.

Animals↗
PubMed2002

Identifying and quantifying sources of variation in microarray data using high-density cDNA membrane arrays.

Microarray experiments involve many steps, including spotting cDNA, extracting RNA, labeling targets, hybridizing, scanning, and analyzing images. Each step introduces variability, confounding our ability to obtain accurate estimates of the biological differences between samples. We ran repeated experiments using high-density cDNA microarray membranes (Research Genetics Human GeneFilters Microarrays Version I) and 33P-labeled targets. Total RNA was extracted from a Burkitt lymphoma cell line (GA-10). We estimated the components of variation coming from: (1) image analysis, (2) exposure time to PhosphorImager screens, (3) differences in membranes, (4) reuse of membranes, and (5) differences in targets prepared from two independent RNA extractions. Variation was assessed qualitatively using a clustering algorithm and quantitatively using a version of ANOVA adapted to multivariate microarray data. The largest contribution to variation came from reusing membranes, which contributed 38% of the total variation. Differences in membranes and in exposure time each contributed about 10%. Differences in target preparations contributed less than 5%. The effect of image quantification was negligible. Much of the effect from reusing membranes was attributable to increasing levels of background radiation and can be reduced by using membranes at most four times. The effects of exposure time, which were partly attributable to variation in the scanning process, can be minimized by using the same exposure time for all experiments.

Algorithms↗