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A Taqman amplicon targeting the nucleocapsid gene of severe acute respiratory syndrome coronavirus (SARS-CoV) is 5 log(10) times more sensitive for SARS-CoV target RNA extracted from infected cells and 2.79 log(10) times more sensitive for RNA extracted from patient material of the index case in Frankfurt than an amplicon targeting the polymerase gene.
Hepatitis A virus (HAV) RNA was extracted from cell culture, serum, liver, and feces and then detected by molecular hybridization with cloned HAV cDNA. Hybridization was approximately 10-fold more sensitive than immune electron microscopy or radioimmunoassay was and less sensitive than was assays of HAV infectivity in primates or in cell culture. As little as 10(3) 50% infective doses of HAV, or approximately 0.1 pg of viral RNA, was detected by this method. Analysis of fecal specimens from an experimentally infected marmoset and an epidemic of hepatitis A showed that HAV excretion could often be detected later in the illness by hybridization than by radioimmunoassay. This technique should be widely applicable for detection and analysis of HAV RNA.
During meiotic maturation and early embryonic cycles, the activity of maturation-promoting factor (MPF) cycles in exact correspondence with the mitotic cycles. For the appearance of MPF activity in starfish, protein synthesis is required except in the first meiotic cycle. In order to identify newly synthesized proteins involved in the regulation of MPF activity, we extracted poly(A)+ RNA from starfish eggs, and found that the egg poly(A)+ RNA induced germinal vesicle breakdown (GVBD) upon injection into immature oocytes of starfish and Xenopus. The molecular size of the poly(A)+ RNA responsible for GVBD was estimated to be approximately 22S by sucrose density gradient centrifugation. Since these characteristics of the starfish egg poly(A)+ RNA are similar to those of cyclin mRNAs from sea urchin and surf clam eggs, we synthesized a 50-mer antisense-cyclin oligonucleotide probe coding for a part of the sea urchin cyclin cDNA and used this to screen starfish RNA. The Northern blot analysis showed that the starfish egg RNA contained cyclin homologous transcripts. Incubation of the starfish egg poly(A)+ RNA and the antisense-cyclin oligonucleotide with RNase H completely destroyed its GVBD-inducing activity. These results indicated that starfish cyclin mRNA was the only poly(A)+ RNA responsible for GVBD. We constructed a starfish egg cDNA library to clone starfish cyclin cDNA. The longest cDNA clone containing 2190 base pairs was sequenced. The longest open reading frame consisted of 395 amino acid residues, and the predicted molecular size was 48 kDa. Comparison of the deduced amino acid sequences of starfish cyclin with known cyclins indicated that the starfish cyclin belongs to the B-type. Injection of synthetic mRNA of starfish cyclin caused GVBD in immature oocytes of starfish and Xenopus, while injection of synthetic mRNA of human CDC2 had no effect. The Northern blot analysis of starfish RNA extracted at various stages of the meiotic cycles suggested that the starfish cyclin transcript was stored in its polyadenylated form even in immature oocytes and was further polyadenylated at maturation.
Analysis of gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Although there are many techniques used to measure gene expression the validity of any such experiment is directly related to the quality of the RNA in the samples. The most readily available source of human brain tissue is post-mortem and while frozen tissue is sometimes available, most archived tissue is fixed and paraffin-embedded. The use of fixed tissue for expression analysis introduces variables, which must be considered in the experimental design. In addition, factors associated with clinical variability of the patient and with tissue procurement can affect RNA transcript levels. In order to illustrate the effects of two common tissue fixatives, formalin and ethanol, on the quality of RNA for expression analysis we compare RNA extracted from these fixed tissues to the gold standard, flash-frozen tissue. We describe RNA extraction from fixed tissue and ways to assess the quality or intactness of the RNA using reverse transcription combined with polymerase chain reaction amplification. An advantage of using archived tissue is the ease with which single cells or subpopulations of cells can be obtained by laser microdissection. The successful isolation of RNA from microdissected cells is also presented. From our results and a review of the literature we conclude that RNA from fixed tissues is a viable source of RNA for expression analysis which should enable new experimental approaches and discoveries as long as attention is given to variables that can affect RNA at all levels of analysis.
A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumin. A usual RNA extraction step yields rRNA accessible for hybridization or reverse transcription-PCR. Hybridization with specific oligonucleotide probes was used for group-specific yield comparison. By using a probe hybridizing to the 16S rRNA of the bacterial kingdom, total bacterial rRNA yield was estimated to be in the range of 0.2 microgram per g for different soils. Group-specific probes did not indicate a selectivity of the isolation procedure and differentiated the compositions of different soil microbial communities. The sequence diversity of the isolated RNA population was also revealed by temperature gradient gel electrophoresis of reverse transcription-PCR amplification products by using a region of the 16S rRNA as a target. The pattern obtained by this analysis differed from a similar one resulting from the separation of amplification products of community DNA preparations. This different view of the community composition is attributable to the correlation of ribosome numbers to the metabolic activity of bacteria in the habitat under observation.
The adenovirus type 5 early region 1B encodes a 55 kd polypeptide that functions after transcription and processing to facilitate cytoplasmic accumulation of late viral mRNAs during lytic infection. A virus, dl338, carrying a deletion within the coding region for the 55 kd product, was found to be cold-sensitive for growth. Accumulation of late viral mRNAs was more severely inhibited at 32 than at 37 degrees C in dl338-infected cells. The metabolism of late viral transcripts was analysed within the nucleus of dl338-infected cells at 32 degrees C. Late viral mRNAs failed to accumulate efficiently within a nuclear compartment defined by specific RNA extraction conditions. Normally, RNA accumulated within this operationally defined compartment after leaving the nuclear matrix and before associating with the nuclear envelope. These results indicate that the 55 kd polypeptide encoded by early region 1B facilitates an intranuclear step in the metabolism of viral mRNAs, resulting in improved transport of these RNAs to the cytoplasm.
We describe here a cDNA:RNA hybridization system for the study of human rhinoviruses. We have constructed an M13 probe from the 5' end of the genome of rhinovirus 14 (HRV-14) and used this to detect directly viral RNA. Of the 56 human rhinoviruses so far investigated 54 or 96.4% gave clearly positive hybridization signals. However, the strength of this signal depended very much on the molecular relationship of these viruses. Thus, HRV-3, 4, 17, 72, and, to a slightly lesser extent, HRV-2, 6, 9, 13, 19, 31, 42, 49, 64, and 69 appear to be closely related to HRV-14 whereas HRV-5, 7, 8, 16, 32, 40, 45, 55, 56, 63, 80, 82, and 85 appear to be relatively divergent. Further, evidence is provided in this study that indicates that it would be feasible to use cDNA probes to detect human rhinoviruses in nasal washings. However, the sensitivity of detection was clearly affected by both the inclusion of inhibitors of endogenous RNase activity in the RNA extraction mixture and also in the method of extracting the viral RNA. From reconstruction experiments in nasal washings and under optimal conditions, we can detect virus at 10(2.8) TCID50/ml.
Dye-encapsulating unilamellar DNA oligonucleotide-tagged liposomes were prepared and characterized for use as signal-enhancing reagents in a microtiter plate sandwich-hybridization analyses of single-stranded RNA or DNA sequences. The liposomes were synthesized using the reversed-phase evaporation method and tagged with DNA oligonucleotides by adding cholesteryl-modified DNA reporter probes to the initial lipid mixture. Liposomes were prepared using probe coverages of 0.0013-0.103 mol% of the total lipid input, several hydrophobic and poly(ethylene glycol)-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 200 nm to 335 nm. Their signal enhancement functionality was compared by using them in microtiter plate sandwich-hybridization assays for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.103 mol%, a liposome diameter of 274 nm, and a phospholipid concentration of 0.3 mM were found. The length between the cholesteryl anchor and the probe was optimal when a spacer composed of TEG+(CH2O)3 was used. Under optimal conditions, a detection limit of 0.5 nM for a truncated synthetic DNA sequence was found with a coefficient of variation of 4.4%. A 500-fold lower limit of detection using fluorescence was found using lysed dye-encapsulating liposomes versus a single fluorescein-labeled probe. Finally, when this method was applied to the detection of atxA RNA extracted from E.coli SG12036-pIu121 and amplified using NASBA, a minimum extracted concentration of RNA of 1.1x10(-7) microg/microL was found.
Radioimmunosorbtion of influenza virus nucleocapsids from lysates of the infected cells was applied for studies on virus-specific RNA synthesis. The method allowed the isolation of intact-labelled viral RNA segments. The procedure included preadsorbtion of antiviral antibodies on protein A containing sorbents. The protein A containing sorbent with attached antibodies was mixed with lysates of influenza virus-infected [3H]uridine-labelled cells. Viral nucleocapsids bound by the antibodies to the sorbent were used for RNA extraction. The isolated RNA was analysed by polyacrylamide gel electrophoresis with subsequent autoradiography. The method allows the isolation of nondegraded labelled virus-specific RNA by means of a relatively simple procedure.
An efficient protocol for the purification of pea seed-borne mosaic potyvirus (PSbMV) particles was developed. This led to the purification of 10 PSbMV isolates by a single procedure. Virus aggregation during purification did not occur and consequently, high virus yield was consistently obtained. The virus thus purified was suitable for preparing viral genomic RNA, although conventional methods for RNA extraction resulted in RNA degradation. An alternative method was adopted which yielded reproducibly full length and infectious RNA. This was applied to three isolates of PSbMV and the RNA used to direct complementary DNA synthesis which in turn yielded nearly full length cDNA products.
BACKGROUND: We evaluated the diagnostic efficacy of urinary angiogenin (ANG) and cytokeratin 20 (CK-20) mRNA in comparison with voided urine cytology in the detection of bladder cancer patients. OBJECTIVES AND METHODS: A total of 97 Egyptian patients provided a single voided urine sample for ANG, CK-20 and cytology before cystoscopy. Of the 97 cases, 63 were histologically diagnosed as bladder cancer; 33 with transitional cell carcinoma (TCC) and 30 with squamous cell carcinoma (SCC), whereas the remaining 34 had benign urological disorders. A group of 46 healthy volunteers were also included in this study. Voided urine was centrifuged and the supernatant was used for estimation of ANG by EIA and confirmed by Western blotting (WB). The urine sediment was used for cytology and RNA extraction. CK-20 RNA was detected by RT-PCR. RESULTS: The best cutoff value for ANG was calculated by a ROC curve as 322.7 ng/mg protein. The median urinary ANG level in bladder carcinoma, benign urological disorders and healthy volunteer groups was: 802.7, 425 and 33 pg/mg protein, respectively. The positivity rate for urinary CK-20 mRNA of the control, benign and malignant groups was 0%, 2.9% and 82.3%, respectively (P = 0.000); while the rates for ANG were 11.6%, 54.8% and 75.4%, respectively (P = 0.000). There was no significant difference in positivity rates of CK-20 and ANG with respect to sex, smoking, schistosomiasis, urine cytology, tumor grade, tumor stage, hematuria or pus cells. The overall sensitivity and specificity were 71.4% and 90% for voided urine cytology, 75.4% and 70.3% for ANG, and 82.3% and 98.8% for CK-20. Combined sensitivity of voided urine cytology with ANG and CK-20 together (98.2%) was higher than either the combined sensitivity of voided urine cytology with ANG (96.5%) or with CK-20 (91.6%) or than that of the biomarker alone. We demonstrated significant positive correlation between CK-20 positivity with age (P = 0.043) and nodal involvement (P = 0.037); however, there was no significant correlation between CK-20 and ANG with the other clinicopathological parameters. CONCLUSIONS: Our data indicate that CK-20 and ANG in voided urine had higher sensitivities compared to voided urine cytology. However, when specificity was considered, CK-20 alone had superior sensitivity and specificity compared to ANG and voided urine cytology.
BACKGROUND: Systemic chemotherapy for operable breast cancer substantially decreases the risk of death. Patients often have de novo resistance or incomplete response to docetaxel, one of the most active agents in this disease. We postulated that gene expression profiles of the primary breast cancer can predict the response to docetaxel. METHODS: We took core biopsy samples from primary breast tumours in 24 patients before treatment and then assessed tumour response to neoadjuvant docetaxel (four cycles, 100 mg/m2 daily for 3 weeks) by cDNA analysis of RNA extracted from biopsy samples using HgU95-Av2 GeneChip. FINDINGS: From the core biopsy samples, we extracted sufficient total RNA (3-6 microg) for cDNA array analysis using HgU95-Av2 GeneChip. Differential patterns of expression of 92 genes correlated with docetaxel response (p=0.001). Sensitive tumours had higher expression of genes involved in cell cycle, cytoskeleton, adhesion, protein transport, protein modification, transcription, and stress or apoptosis; whereas resistant tumours showed increased expression of some transcriptional and signal transduction genes. In leave-one-out cross-validation analysis, ten of 11 sensitive tumours (90% specificity) and 11 of 13 resistant tumours (85% sensitivity) were correctly classified, with an accuracy of 88%. This 92-gene predictor had positive and negative predictive values of 92% and 83%, respectively. Correlation between RNA expression measured by the arrays and semiquantitative RT-PCR was also ascertained, and our results were validated in an independent set of six patients. INTERPRETATION: If validated, these molecular profiles could allow development of a clinical test for docetaxel sensitivity, thus reducing unnecessary treatment for women with breast cancer.
Hepatitis A is one of the major infectious diseases epidemiologically associated with worldwide shellfish consumption. Molecular detection using polymerase chain reaction (PCR) to detect hepatitis A virus (HAV) in contaminated shellfish can be hindered by low virus recoveries during the concentration process and by natural PCR inhibitors in shellfish. This study evaluated and modified two major steps of a processing procedure for virus concentration from oysters: acid adsorption-elution and solvent extraction. With the addition of second and third elutions, the acid adsorption-elution step doubled the recovery to 46% of HAV seeded initially. Extraction with chloroform or chloroform-butanol resulted in lower HAV detection limits by reverse transcription-PCR (RT-PCR)-oligoprobing than extraction with the fluorocarbon, Freon. These results led to the following modified procedure: HAV was acid adsorbed at pH 4.8, eluted first with 0.05 M glycine, second with 0.5 M threonine, PEG-precipitated twice, chloroform-extracted twice, RNA-extracted, and RT-PCR (single round) amplified. Using the modified procedure, HAV was detected by RT-PCR in all trials with a seeding density of > or = 1 plaque forming unit (PFU)/g of oyster, and in which the equivalent detection limit was 0.33 PFU of HAV seeded per RT-PCR reaction (corresponding to 111 PCR units). The method developed is capable of detecting low levels of HAV in oysters environmentally contaminated.
OBJECTIVE: To explore the patterns of TCR V beta gene families and the preferentially used V beta genes in intrathyroidal T lymphocytes (ITL) of patients with Graves disease and Hashimoto thyroiditis. METHODS: Thyroid tissues of 12 patients with Graves disease, and 15 patients with Hashimotos thyroiditis were used for RNA extraction. Then, the RNA was used as templates for RT-PCR with a panel of primers including 24 oligonucleotides specific for TCR V beta gene families as upstream primers and one oligonucleotide specific for C beta as downstream primer. The amplified products were subjected to electrophoresis in agarose gels and observed under UV light. The positive expression of TCR V beta gene was identified by comparing the products with the size of the designed V beta genes. The matched peripheral blood samples of 5 Graves patients, 5 Hashimoto thyroiditis patients and 7 normal subjects were also obtained and used as controls. RESULTS: In 24 TCR V beta gene families tested for each individual sample, their average expressions were 5.3 +/- 1.2 and 13.4 +/- 3.0 in thyroid specimens of patients with Graves disease and Hashimoto thyroiditis, respectively, while those in the matched peripheral blood lymphocytes of the above two groups and normal controls were 23.0 +/- 1.0, 22.2 +/- 1.3 and 22.4 +/- 1.7, respectively. In addition, some of the TCR V beta genes such as V beta 3, V beta 5 and V beta 8 were more frequently used by intrathyroidal lymphocytes of Graves disease. CONCLUSIONS: These data indicate the oligoclonality of the intrathyroidal T cell populations in patients with Graves disease. Also, those of the preferentially used TCR V beta genes can be regarded as molecular targets for specific immune intervention. In contrast, the loss of restriction of TCR V beta gene utilization exists in Hashimoto thyroiditis, which may be related to the recruitment of T lymphocytes in thyroid glands through this mechanism.
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A novel method for the extraction of HCV-RNA by using DNA probe coated-latex microparticle (AmpliTex) was developed. This high density particle, with a large surface area, could be dispersed uniformly in a guanidium lysate of the serum. The DNA probe on the particles could hybridize with a few copies of HCV-RNA in the lysate. The RNA thus extracted on the particle can be subjected to the reverse-transcription and PCR reaction. Analytical sensitivity to detect HCV in serum was approximately 20 copies per ml. AmpliTex and the conventional organic extraction gave the same sensitivity and specificity (both 100%) to detect the HCV-RNA from fifty clinical specimens. It was also possible to extract the HCV-RNA from heparinated plasma.
The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a "gold standard" reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of >or=1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples.
The antinociceptive potency of the delta opioid receptor (DOR) agonist [D-Ala2]Deltorphin II and the levels of DOR mRNA were measured in mice chronically treated with naltrexone. ED50 determinations for [D-Ala2]Deltorphin II, using the tail-flick test with mice that had been treated with naltrexone for 7 days followed by a 24 h naltrexone free period (study day 8), revealed a 7.7-fold increase in antinociceptive potency, indicating functional supersensitivity. Utilization of a micro-dissection technique followed by quantitative solution hybridization measurements of RNA extracts from mouse CNS revealed levels of DOR mRNA ranging from 2.8 pg/microgram RNA in the caudate-putamen to 0.3 pg/microgram RNA in cerebellum. However, despite the functional increase in DOR sensitivity, the DOR mRNA levels in selected brain areas and spinal cord of naltrexone-treated and control mice did not differ. Assessment of DOR mRNA levels in whole brain and selected CNS regions after shorter treatment intervals (1, 6 and 12 h and 2 and 7 days) in naltrexone-treated and control mice revealed a similar pattern of results. Northern blot analysis of mouse whole brain RNA extracts after 7 days of naltrexone treatment did not show any alteration in the size of the DOR transcript. These data demonstrate that DOR mRNA levels are not altered during and after chronic naltrexone treatment and therefore are not associated with opioid-induced DOR up-regulation or DOR functional supersensitivity.