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PubMed1981

Purification of human lymphoblastoid cell-derived interferon-alpha by controlled-pore glass bead adsorption chromatography and molecular sieving.

Human lymphoblastoid cell-derived interferon-alpha has been purified by a combination of chromatography on controlled-pore glass 350, gel filtration on Ultrogel AcA 54 and hydrophobic chromatography on Phenyl-Sepharose CL-4B. Specific activities of greater than 10(7) units/mg have been obtained. Controlled-Pore Glass has the advantages of accommodating large volumes of induced medium in an "in-line" process giving high recoveries of interferon and removing most of any contaminating DNA.

Cells, Cultured↗
PubMed1990

Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells.

Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low-abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co-reverse-transcribed and co-amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments. Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.

Animals↗
PubMed2006

Human-blind probes and primers for dengue virus identification.

Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe 'host-blind probe design'- a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is approximately 1 : 10(150).

DNA Primers↗
PubMed2003

Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR.

We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.

Adenoviruses, Human↗
PubMed2003

mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil.

The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH(4)NO(3) were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r(2) = 0.72) and in liquid culture (r(2) = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.

Azotobacter vinelandii↗
PubMed2006

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

Histidine↗
PubMed1978

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth.

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.

Adenoviridae↗
PubMed1993

Incorporation of 5-fluorouracil into RNA by Mycobacterium avium complex strain LM1.

The incorporation of 5-fluorouracil (FUra) into RNA of Mycobacterium avium complex strain LM1 was evaluated. Cells were labeled with either [14C]FUra or [3H]uracil and the ribonucleosides were analyzed by high-performance liquid chromatography. The identification of the ribonucleosides was facilitated by the use of an isocratic system that provided unambiguous identification of the RNA pyrimidine components. Uracil was incorporated into RNA as uridine, but an equal amount was converted to cytidine. [14C]FUra was incorporated directly into RNA as 5-fluorouridine and there was no evidence of its conversion to other pyrimidines. The ratio of 5-fluorouridine:uridine was 2.8-fold greater for cells grown in 100 micrograms FUra/mL than for cells grown in 20 micrograms FUra/mL. Analysis of the RNA nucleotides was performed and deoxyribonucleotides were present; DNA contamination was estimated to range from about 2 to 8% of the RNA preparations.

Biotransformation↗
PubMed2000

High percentage of false-positive results of cytokeratin 19 RT-PCR in blood: a model for the analysis of illegitimate gene expression.

Cytokeratin 19 (CK19) RT-PCR is widely used in order to detect circulating tumor cells in peripheral blood and bone marrow. However, increasing amounts of information support the fact that it is also associated with a high percentage of false-positive results. In our study, we not only managed to demonstrate the significant limitations of this method, but were also able to clarify the reasons behind these limitations. We developed a completely novel RT-PCR for CK19 and sequenced an intron at nucleotide (nt) 980 of the CK19 mRNA to exclude DNA contamination. Tumor dilution experiments were performed in order to analyze the specificity and sensitivity of the method. Control experiments using the blood of healthy donors were performed. Tumor cell dilution experiments gave a detection limit of one tumor cell. If tumor cells were mixed with an equal volume of pure mononuclear cells, the detection limit was 1 tumor cell in 10(5) mononuclear cells. RT-PCR of mononuclear cells from healthy blood donors gave false-positive results in 29% of the cases. We conclude that a significant decrease in the sensitivity of CK19 RT-PCR occurs if it is performed in blood cells and that the illegitimate CK19 gene expression in normal cells can lead to false-positive results. These limitations have to be taken into account if RT-PCR is to be used for the detection of tumor cells either in blood or in bone marrow in clinical practice.

Blood Donors↗
PubMed2006

The association between telomerase activity and expression of its RNA component (hTR) in breast cancer patients: the importance of DNase treatment.

Telomerase is a ribonucleoprotein enzyme that compensates for the telomere length shortening which occurs during the cell cycle. Telomerase activity has been detected in most tumours but not in somatic cells. However, hTR; the RNA component of telomerase; has been reported to be universally expressed in both cancerous and non-cancerous tissues. Tumour samples from 50 patients with primary invasive breast cancer were collected. The TRAP assay was used to detect telomerase activity. RT-PCR on cDNA and DNased cDNA samples and control groups was used to detect the expression of hTR, GAPDH and PGM1 genes. Seventy-two percent of samples showed telomerase activity. DNA contamination was detected in 36 (72%) of RNA samples. Without performing DNase treatment, 49 (98%) of all samples showed hTR expression, but with the application of this strategy, hTR expression decreased from 98% to 64%. A significant association (p < 0.001) between hTR expression and telomerase activity was observed. Among the 32 hTR positive samples, 30 had telomerase activity and among the 18 hTR negative samples, telomerase activity was observed in 6 cases. Thus the application of this strategy could provide an applicable tool to use instead of the TRAP assay thus facilitating telomerase research in cancer genetic investigations.

Journal Article↗
PubMed1994

Detection of placental growth hormone variant and chorionic somatomammotropin ribonucleic acid expression in human trophoblastic neoplasms by reverse transcriptase-polymerase chain reaction.

Attempts to assess human placental GH variant (hGH-V) and chorionic somatomammotropin (hCS) RNA in choriocarcinoma cell lines have been hampered by low levels of expression and limited sensitivity of RNA blotting analysis. We examined human choriocarcinoma BeWo, JAR, and JEG-3 cell lines as well as samples of complete hydatidiform moles for expression of members of the human GH (hGH) gene family using reverse transcriptase-polymerase chain reaction. A single and common set of primers was designed and used to detect products of the hGH/hCS genes as well as distinguish processed RNA from any contaminating DNA. Transcripts from the hCS genes hCS-A and -B were distinguished from placental hGH variant (hGH-V) and hCS-like (hCS-L) gene RNA by diagnostic restriction digestion of the polymerase chain reaction products. The expected pattern of hGH/hCS RNA expression was detected in term placenta, where hCS and hGH-V/hCS-L transcripts represented approximately 95% and approximately 5% of the total hGH/hCS RNA, respectively. The level of hCS RNA varied from 22-99% of the total hGH/hCS RNA in the neoplastic trophoblast samples, and variable levels of hGH-V and hCS-L RNA were also observed. In choriocarcinoma JAR cells, hGH-V RNA represented approximately 78% of the total hGH/hCS RNA compared to approximately 22% for hCS. Further, although low hCS-L RNA levels (< 1%) were found in term placenta and two of the hydatidiform moles, hCS-L transcripts represented 11% of the total hGH/hCS RNA in a third hydatidiform mole. Finally, in contrast to the detection of variable levels of hCS-L RNA in term placenta and hydatidiform mole samples, no hCS-L transcripts were detected in the three choriocarcinoma cell lines examined. These patterns reflect either deregulated hGH/hCS gene expression in neoplastic trophoblasts or differences that accompany the process of differentiation of trophoblast subpopulations. Regardless, this suggests that the control of hGH-V and hCS-L gene expression is distinct from that of the hCS-A and hCS-B genes and raises questions about the possible involvement of hGH/hCS family members in the pathology of placental abnormalities.

Base Sequence↗
PubMed2005

Extraction of RNA from cells and tissue.

Total undegraded cellular RNA, largely free of contaminating DNA, can be rapidly and easily isolated from homogenized tissue or cultured cells using acid pH guanidinium thiocyanate/phenol/chloroform extraction. Commercially available acid phenol/guanidinium thiocyanate solutions allow one-step complete solubilization of tissue or cells under conditions that inhibit RNAse activity. We describe here a detailed procedure for total RNA extraction from homogenized tissues or cultured cells using such reagents, together with additional purification steps by passage through Phase-Lock gel tubes, and recommend optional chromatographic poly(A+) RNA enrichment procedure using Mini Oligo(dT) spin columns, together with the spectrophotometric determination of RNA yield and purity.

Cells, Cultured↗
PubMed2006

Diagnosis and monitoring of chronic myeloid leukemia by qualitative and quantitative RT-PCR.

Real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) methods for the quantitation of BCR-ABL mRNA in the blood of patients with chronic myeloid leukemia (CML) has become the predominant molecular monitoring technique. The BCR-ABL fusion gene is expressed in over 95% of patients with CML, and RQ-PCR provides a reliable, high-throughput method to accurately assess the level of treatment response and provides an early indication of emerging drug resistance. The ABI Prism 7700 Sequence Detection System uses TaqMan fluorogenic probes to quantitate specific nucleic acid sequences using RQ-PCR. The analyzer monitors an increase in fluorescence during the PCR cycle, which is proportional to the amount of accumulated product. The starting copy number is calculated relative to a series of standards. The copy number is normalized to a control gene that compensates for variations in the efficiency of the RT step and for the degree of RNA degradation. In our experience, reliable and consistent RQ-PCR requires thorough validation of all aspects of the procedure, including the selection of an appropriate control gene, careful assay design to avoid polymorphisms in primer or probe binding sites and to exclude the amplification of contaminating DNA, and monitoring the performance of the RQ-PCR by the use of quality control samples.

Cloning, Molecular↗
PubMed2001

Small-sample total RNA purification: laser capture microdissection and cultured cell applications.

Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR.

3T3 Cells↗
PubMed1999

[Establishment of studying system cloned vernalization-putative cDNA clone by means of differential display PCR].

Differential display PCR has been developed as a tool detecting and characterizing specially expressed gene in eukaryotic cells recently. In this paper, a studying system was established which applied the novel approach of differential display PCR to clone vernalization-related cDNA clone. Here methodological details were provided which included the purification of total RNA, removal of contaminated DNA, reverse transcription of cDNAs, parameters of PCR, electrophoresis of amplified cDNAs, recovery and reamplification of cDNAs. It was also identified a vernalization-putative cDNA clone (VPC) of VPC28 which was only expressed at the key stage of vernalization for 20 d in winter wheat. These results moreover made it readily applicable to a broad spectrum of similar studies.

Cloning, Molecular↗
PubMed2002

[Screening of stage-specific expression genes of Spirometra erinacei-europaei plerocercoid by mRNA differential display technique].

OBJECTIVE: To screen stage-specific expression genes of plerocercoid of Spirometra erinacei-europaei. METHODS: RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform from plerocercoid and adult worm of Spirometra erinacei-europaei. Contaminated DNA in the RNA was digested by RNase-free DNase. cDNA was synthesized by using T12MA, T12MC, T12MG and T12MT primers, and PCR was then done using the same T12MN and one random primers. The PCR products were fractionated on 8% denatured polyacrylamide gel, differential bands of plerocercoid found in the gel were cut out, amplified by PCR and sequenced after the gel was dried out and autoradiographed. Northern hybridization was conducted to identify the stage-specific expression genes. RESULTS: Eleven differential bands were selected from the gel and classified into 3 kinds of gene fragments by hybridization after they were amplified by PCR. The fragments 1 and 2 were confirmed to express specifically in plerocercoid by Northern hybridization, but the fragment 3 was expressed in both plerocercoid and adult worm. When the 3 gene fragments were homologically analyzed in GenBank, the sequence which was homologous with the fragments 1 and 2 was not found, but the fragment 3 had high homology with many kinds of 28S rRNA. CONCLUSION: The gene expression of plerocercoid was different from that of adult worm probably because they live in different hosts. Two kinds of different gene fragments in plerocercoid were identified by mRNA differential display technique.

Amino Acid Sequence↗
PubMedDate not supplied

Selection of cDNAs for phosphodiesterases that hydrolyze guanosine 3';5'-monophosphate in Escherichia coli.

A genetic selection procedure has been developed which makes the growth of E. coli dependent on expression of a cGMP phosphodiesterase cDNA. E. coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP. If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source. The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly. Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E. coli guaA auxotroph in the presence of cGMP. Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.

3',5'-Cyclic-GMP Phosphodiesterases↗