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PubMed2005

Detection of disseminated lung cancer cells in regional lymph nodes by assay of CK19 reverse transcriptase polymerase chain reaction and its clinical significance.

PURPOSE: To set up a molecular method (i.e. RT-PCR) that can be used to detect disseminated tumor cells (DTCs) in regional lymph nodes (LNs) in patients with lung cancer and to evaluate its clinical significance. METHODS: Cytokeratin 19 (CK(19)) was used as marker. Serial dilution study for LC-5 cells (a lung squamous cell line) was performed to detect sensitivity of the molecular protocol. Regional LNs (n = 261) and primary lung cancer tissue (n = 40) were obtained from 40 patients with lung cancer who underwent lobectomy or pneumonectomy. They were randomly categorized into two groups: group I (LN-based study, n = 20) and group II (patient-based study, n = 20). Each LN was halved. One half of a LN was subjected to histological examination (HE) and the other half was subjected to RT-PCR amplification of CK(19) mRNA. The effect on survival was analyzed. The cumulative survival was calculated by the Kaplan-Meier method and compared by the log rank test. The Cox model analyzed the prognostic factors. RESULTS: CK(19) mRNA expressed in all tumor tissues as well as LC-5, PAa cells (a lung adenocarcinoma cell line), but not in normal control LNs. Serial dilution study for LC-5 cells demonstrated that CK(19) mRNA was detectable at a concentration as low as 10 LC-5 cells in 1x10(7) LN cells. There was no significant difference between the detecting result of single LN and that of mixed LNs (P > 0.05). In 18 of 40 patients, the metastasis in regional LNs was found by both HE and RT-PCR. Of 22 patients without pathologically involved nodes, six (27%) were found to express CK(19) mRNA in regional LNs. According to the results of regional LNs in 40 patients by molecular assay, the presence of the CK(19) product in LNs was related to tumor size (chi(2) = 5.76, P < 0.025) as well as cell differentiation of the tumor (chi(2) = 7.08, P < 0.01). Following a median observation time of 26 months (range, 4 to 60 months), patients with DTCs in nodes showed significant shorter disease-free survival duration than node-negative patients (log-rank test, P = 0.001). The independence of this prognostic significance was demonstrated by a multivariate analysis (Cox regression model, P = 0.004). The results diagnosed by HE had no significant effect on prognoses (P = 0.455). CONCLUSIONS: Comparing with HE, RT-PCR can make more accurate assessment of metastatic status in LNs, which is helpful for screening the patients in whom the early subclinical metastasis exists and disclosing the intrinsic regulation of malignant metastasis. The presence of DTCs in LNs is an independent factor for prognosis. Molecular detection of DTCs in LNs is a supplement for current tumor staging in lung carcinoma.

Aged↗
PubMed2000

Differences in IgE isotype switching induced by anti-CD40 monoclonal antibody and cytokines among subtypes of chronic B lymphoid leukemias.

OBJECTIVE: Immunologic differences among the chronic B lymphoid leukemias defined by the French-American-British (FAB) classification were evaluated with respect to IgE isotype switching induced by anti-CD40 monoclonal antibody (mAb) and cytokines. MATERIALS AND METHODS: We immunocytochemically studied IgE isotype switching of leukemic B cells from 25 cases and three cell lines established from the leukemias after stimulation with anti-CD40 mAb, plus each of the following cytokines: interleukin 2 (IL-2); IL-4; IL-10; tumor necrosis factor alpha (TNF-alpha); and transforming growth factor beta (TGF-beta). Also, genomic Cepsilon and Cepsilon transcripts were analyzed by polymerase chain reaction and reverse transcriptase polymerase chain reaction. RESULTS: Leukemic cells from hairy cell leukemia variant and its cell line, with deletion of the Cepsilon gene, did not undergo IgE isotype switching in response to any of the stimuli. In contrast, a cell line (FH-5) established from chronic lymphocytic leukemia cells, bearing the Cepsilon gene, underwent the highest level of IgE isotype switching on stimulation with anti-CD40 mAb and IL-4. This response was correlated with the production of Cepsilon transcripts. IL-4, IL-10, and TNF-alpha induced higher levels of IgE isotype switching than the others. No IgE isotype switching was observed in any of the non-Hodgkin's lymphomas examined, except mantle cell lymphoma and lymphoplasmacytic lymphoma. Percentages of CD40(+) cells in five cases with follicular lymphoma were significantly lower than the other leukemias. CONCLUSIONS: IgE isotype switching induced by anti-CD40 mAb with cytokines other than IL-4 was first demonstrated, whereas none of the non-Hodgkin's lymphomas except mantle cell lymphoma and lymphoplasmacytic lymphoma showed IgE isotype switching in response to any of the stimuli. Cells of follicular lymphoma were suggested to be different from cells of the other leukemias.

Antibodies, Monoclonal↗
PubMed2005

Identification of clonotypic IgH VDJ sequences in multiple myeloma.

In multiple myeloma (MM) the rearranged immunoglobulin heavy chain (IgH) variable, diversity, and joining (VDJ) DNA sequence of malignant plasma cells (PCs) serves as a marker for cells in the MM clone. This clonotypic sequence can be isolated from MM PCs by reverse transcriptase polymerase chain reaction (RT-PCR) with consensus primers that amplify the rearranged IgH repertoire. This chapter focuses on the key steps in determining patient-specific clonotypic sequences, including bulk RT-PCR using purified bone marrow mononuclear cell (BMMC) RNA, single-cell RT-PCR using RNA from PCs sorted by flow cytometry, IgH sequence alignments using IMGT or V BASE, and patient-specific primer design. In a test panel of several MM patient BMMCs, primers specific for the proposed sequence must amplify IgH from only the original patient. Furthermore, the proposed IgH sequence is not confirmed as clonotypic until these primers generate positive amplifications in the majority of single PCs from the original patient. This two-part test ensures that the proposed IgH sequence satisfies the definition of the clonotypic sequence as the most frequent, unique IgH sequence in an MM patient PC sample. With this patient-specific MM marker, a better understanding of transformed PCs and their B-lineage predecessors can be developed.

Base Sequence↗
PubMed1999

Progression of renal disease in interleukin-4 transgenic mice: involvement of transforming growth factor-beta.

Recent reports have suggested the involvement of interleukin-4 (IL-4) in glomerular pathophysiology. Using immunohistochemistry and reverse transcriptase polymerase chain reaction we investigated the renal lesions in transgenic (tg) mice with widely distributed IL-4 expression including the kidney, and measured the serum levels of the cytokines transforming growth factor-beta (TGF-beta) and IL-4 by ELISA. Transgenic animals exhibited glomerular hypertrophy with progressive mesangial sclerosis leading to renal failure. Renal IL-4 transcript expression, mesangial accumulation of collagen types I, III, IV and V, and immune deposition accompanied by increased expression of TGF-beta1 protein and mRNA were observed. Seven day-old transgenic animals showed early renal fibrotic changes in the absence of immune deposits or TGF-beta1 upregulation. The sera of transgenic mice not only showed elevated levels of circulating IL-4 (tg: 76.6 pg/ml +/- 7.1 vs wildtype (wt): < 3 pg/ml), but significantly decreased TGF-beta1 levels (tg: 18.9 ng/ml +/- 4.1 vs wt: 38.7 ng/ml +/- 2.9; P < 0.005). The disease severity correlated with the serum IL-4/TGF-beta1 ratio rather than with the IL-4 concentration. These data suggest that renal IL-4 production results in matrix accumulation prior to any immunological insult, that increased circulating IL-4/TGF-beta1 ratios are associated with renal immunopathological manifestations and that upregulation of renal TGF-beta1 expression following glomerular Ig deposition accelerates the sclerosis and exacerbates disease development.

Animals↗
PubMed2002

Very low frequencies of human normal CD34+ haematopoietic progenitor cells express the Wilms' tumour gene WT1 at levels similar to those in leukaemia cells.

The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1-expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single-cell reverse transcriptase polymerase chain reaction (RT-PCR) methods. Using the semiquantitative single-cell polyA-PCR + sequence-specific (SS)-PCR method, WT1 expression was detected in four (1.3%) out of 319 CD34+ BM single cells. To confirm the above results, a single-cell nested sequence-specific (NSS)-RT-PCR method that was less quantitative but more sensitive than the polyA-PCR + SS-PCR method was also performed, and WT1 expression was detected in 15 (1.1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1.2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1-expressing cells were found between CD34+CD38- and CD34+CD38+ BM single cells. Furthermore, WT1-expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage-specific and cell cycle gene expression in WT1-expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+CD38- and lineage-committed, proliferating CD34+CD38+ BM cells. Our results could indicate that these WT1-expressing CD34+ BM cells were normal counterparts of leukaemia cells.

Antigens, CD34↗
PubMed2006

Immunoglobulin genes in multiple myeloma: expressed and non-expressed repertoires, heavy and light chain pairings and somatic mutation patterns in a series of 101 cases.

BACKGROUND AND OBJECTIVES: The available data on the immunoglobulin gene (IG) repertoire in multiple myeloma (MM) derive mainly from heavy chains; considerably less is known about light chains. We assessed in parallel IGH and IGK/IGL rearrangements in 101 MM patients so as to gain insight into: (i) IG repertoires; (ii) antigen impact; (iii) the role of receptor editing. DESIGN AND METHODS: Bone marrow aspirates were collected from all cases. IGHV-(D)-J and IGLV-J rearrangements were amplified by reverse transcriptase polymerase chain reaction (PCR). In all cases, IGKV-J rearrangements were analyzed in parallel on cDNA/genomic DNA. IGKV-KDE and IGKJ-C-INTRON-KDE were also amplified by DNA-PCR. RT-PCR products were directly sequenced. RESULTS: IGHV3 genes predominated; the IGHV4-34 gene was used in only one case. Five IGKV and five IGLV genes accounted for the majority of in-frame, transcribed IGKV-J or IGLV-J rearrangements. Taking IGKV-J, IGKV-KDE and IGKJ-C-INTRON-KDE rearrangements together, biallelic IGK locus rearrangements were detected in 22/43 k-MM cases. In l-MM, 36/42 cases had at least one rearranged IGK allele; 8/19 IGKV-J rearrangements in l-MM were in-frame. All in-frame, transcribed IGH/IGK/IGL sequences were mutated; parallel heavy/light chain analysis demonstrated a comparable impact of somatic hypermutation. INTERPRETATION AND CONCLUSIONS: Biases in IG repertoire did not seem disease-related but followed a similar pattern to that of the normal repertoire. The under-representation of the IGHV4-34 gene provides an explanation for the paucity of autoimmune phenomena in MM. Somatic mutation patterns indicate the complementary role of MM IGH/IGK/IGL sequences in antigen recognition. Finally, the frequent inactivation of productive IGKV-J joints by secondary rearrangements in MM suggests active receptor editing.

Bone Marrow Cells↗
PubMed2002

Potential mouse tumor model for pre-clinical testing of mage-specific breast cancer vaccines.

Currently, there is a lack of suitable pre-clinical mouse models for testing and optimization of experimental cancer vaccines. Here, in situ developed mammary tumors of MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and normal mammary, liver, spleen, and testis were screened for expression of tumor-associated antigens (TAA) Mage-b1/2/3 by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot hybridization. Mage-b1/2/3 are homologues of the human TAA MAGE-B1/2/3. Expression of these human MAGE genes has been found in tumors of various histological types, including breast cancer. Mage-specific RT-PCR products (using primers that amplify all three Mage-b1/2/3) were detected in mammary tumors of the MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and in testis, but not in other normal tissues. RT-PCR products obtained from the mammary tumors (using primers that amplify the complete protein-encoding region of Mage-b1/2/3) were cloned and sequenced, and appeared to be most homologous with Mage-b3. Comparison of the Mage-b3 gene in mammary tumors and normal tissues suggest that somatic mutations did not occur in the Mage-b3 gene of the ras- and myc-induced mammary tumors. In addition, no differences were found between the Mage-b3 cDNA of testis, the only normal tissue that expresses Mage-b3, and Mage-b3 in genomic DNA of normal kidney, where Mage-b3 is silent. The MMTV-v-Ha-ras and MMTV-c-myc transgenic mice of this study are the first immune competent mouse models with in situ developed mammary tumors in which the expression of Mage-b3 TAA has been demonstrated. This makes them potentially suitable as a mouse model for pre-clinical testing of Mage-specific cancer vaccines in vivo.

Animals↗
PubMed1992

[Detection of minimal residual disease using reverse transcriptase polymerase chain reaction technique].

Polymerase chain reaction (PCR) is a highly sensitive technique to detect minimal residual disease (MRD) of hematological malignancy by amplifying tumor specific nucleotide sequences. When breakpoint is located over large lesions of genomic DNA, like t(9;22) leukemia, PCR amplifying cDNA of chimeric mRNA, called reverse transcriptase PCR (RT-PCR), can be utilized. We applied this method for the detection of MRD in patients with t(1; 19) acute lymphoblastic leukemia. RT-PCR detection of MRD in patients with leukemia might be useful for estimating of the depth of remission, for disclosing preclinical relapse, and for evaluating the efficacy of in vitro purging in autologous bone marrow transplantation.

DNA, Neoplasm↗
PubMed1995

Characterization of the mode of Hantaan virus infection in adult mice using a nested reverse transcriptase polymerase chain reaction: transient virus replication in adult mice.

A polymerase chain reaction (PCR) for the detection of hantavirus genome was established and applied to analyze the mode of infection of Hantaan virus in adult ICR mice. The cDNA for the S genome segment of Hantaan virus was reverse-transcribed from the total RNA of organs of the infected mice. The sequence in the S genome segment of Hantaan virus was successfully amplified by reverse transcriptase (RT)-PCR followed by nested PCR. In 5-week-old ICR mice inoculated intraperitoneally with Hantaan virus, strain 76-118 (1.3 x 10(5) FFU/mouse), the virus was detected in clots and lungs from 3 to 10 days post-inoculation (p.i.) by nested PCR and virus-isolation techniques. No virus was detected in any specimens collected on 1 day and after 28 days p.i., and in spleens and brains through the observation period by both methods. The antibody which was measured by indirect immunofluorescence antibody assay (IFA) appeared at 7 days p.i. and the geometric mean titer was elevated to its maximum level of 1:203 at 10 days p.i., maintaining the same level until 35 days p.i. These results suggest that adult mice are transiently infected with Hantaan virus.

Animals↗
PubMed1995

Separation and quantitation of reverse transcriptase polymerase chain reaction fragments of basic fibroblast growth factor by capillary electrophoresis in polymer networks.

In human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription-polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by slab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P-labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.

Adrenal Cortex↗
PubMed1994

Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction.

At the time of human embryo implantation, large numbers of maternal CD56brightCD16- NK cells appear in the uterus. These unusual lymphocytes are believed to control the migration and differentiation of highly invasive fetally derived trophoblast cells, which infiltrate into the maternal uterus to remodel the spiral arteries during the first trimester. One possible mechanism of control is by cytokine production. In this study, highly purified (> 99%) populations of first trimester decidual CD56brightCD16- NK cells and CD3+ T lymphocytes were obtained by using a FACS. These cells were examined by reverse transcriptase PCR for their expression of mRNAs for the following cytokines: granulocyte-macrophage (GM)-CSF, CSF-1, TNF-alpha, IFN-gamma, TGF-beta 1, leukemia-inhibitory factor (LIF), and IL-2. Then, the expression was compared with that of resting PBL. The identity of the PCR products was verified by Southern blotting and hybridization with cytokine-specific probes. Both decidual CD56brightCD16- NK cells and CD3+ T cells were found to express mRNA for CSF-1, TNF-alpha, IFN-gamma TGF-beta 1, and LIF, but GM-CSF mRNA was detected only in CD56bright NK cells. IL-2 mRNA was detected in only some decidual T cell samples, and then only after at least two rounds of amplification. In contrast, peripheral blood CD56brightCD16- NK cells, CD56dimCD16+ NK cells, and CD3+ T cells expressed mRNA only for TNF-alpha and TGF-beta 1, but not for GM-CSF, CSF-1, IFN-gamma, LIF, or IL-2. These results suggest that both decidual NK cells and decidual T cells produce a variety of cytokines that may be involved in the control of trophoblast migration and differentiation during pregnancy.

Antigens, CD↗
PubMed1995

Early detection of minimal residual disease by reverse transcriptase polymerase chain reaction predicts relapse in acute promyelocytic leukemia.

The PML/RAR alpha fusion RNA can be detected in acute promyelocytic leukemia (APL), cytogenetically characterized by the translocation t(15;17). Our study included ten newly diagnosed patients with APL who were investigated during the course of their diseases using reverse transcription polymerase chain reaction (RT-PCR). At diagnosis, aberrant fragments with a size heterogeneity due to alternative spliced products were detected in all patients, we observed breakpoints within bcr3 (short type) in two patients and bcr1 and 2 breakpoints (long type) in eight patients. Treatment consisted of all-trans retinoic acid (ATRA) in all patients; six patients received simultaneous cytostatic therapy during remission induction. At the time of complete hematological remission (CR), only two patients showed a negative RT-PCR result; eight of the ten patients were still PCR positive when nested primers were used. Subsequently, eight patients received consolidation chemotherapy and became PCR negative. Seven of eight patients are in continuous complete remission (median remission duration: 21 months, range: 11+ -26+ months). One patient of the chemotherapy group became PCR positive after 4 months in complete remission and relapsed after 6 months. The remaining two patients who were treated only with ATRA relapsed, received induction chemotherapy, and are in second and third complete remission, respectively. In conclusion. PCR negativity can be achieved only by chemotherapeutic consolidation; patients treated with ATRA alone remain PCR positive. Relapse is always preceded by a positive PCR result. Surprisingly, also patients without measurable PML/RAR alpha-mRNA in sequential analyses after cytostatic treatment became PCR positive and experienced relapse.

Adult↗
PubMed1995

Fetal cytokine expression in utero detected by reverse transcriptase polymerase chain reaction.

Cytokine expression at the maternal-fetal interface is well documented. Some authors have postulated the existence of a bidirectional cytokine signaling mechanism that is critical to the maintenance of normal pregnancy. The role of the fetus (versus fetally derived placental tissues) in this process is unknown. Using reverse transcription polymerase chain reaction techniques, we studied paired maternal and fetal samples from 16 pregnancies (including two twin pregnancies) for the presence of tumor necrosis factor-alpha and IL-1 beta mRNA. We demonstrate that mRNA for both of these cytokines can be detected in both maternal and fetal blood as early as the 21st wk of gestation. These results support a potential role for the fetus in the bidirectional cytokine signaling of pregnancy.

Base Sequence↗
PubMed1997

Demonstration of estrogen receptor mRNA in bone using in situ reverse-transcriptase polymerase chain reaction.

Falling estrogen levels affect the female skeleton profoundly. Following menopause, estrogen lack is a major cause of osteoporosis. The site of estrogen action in human bone, however, is unclear, but responsive cells must express the estrogen receptor (ER). One obstacle to localizing these cells is that mRNA for ER is expressed in low copy number. Hence, conventional molecular techniques are either too insensitive to detect receptor transcripts (in situ hybridization) or necessitate amplification of RNA extracted from tissue [Northern analysis and polymerase chain reaction (PCR)], thus failing to identify the specific target cells within the mixed-cell population of bone. In situ PCR (IS-PCR) is a technique that combines the sensitivity of PCR with the localization of conventional in situ hybridization. The technique has previously been used primarily to detect single-copy genes and viral DNA within cells. More recently, incorporation of a reverse-transcriptase reaction (IS-RT-PCR) has allowed the technique to be used to identify rare mRNAs within tissues. We have therefore applied the technique of IS-RT-PCR to localize ER mRNA first in human breast tumors, a known positive tissue, and then in bone. Using conventional riboprobe in situ hybridization, ER transcripts were not detectable in any bone cells within sections taken from normal bone and several actively remodeling bone tissues, namely, Paget's disease, renal hyperparathyroidism, and healing fracture callus. The technique of IS-RT-PCR, however, allowed amplification of transcripts to a detectable level. Following two cycles of amplification, hybridization signal was observed in osteoblasts and to a lower level in osteoclasts and occasional osteocytes. This positive signal was more obvious after five cycles, particularly in osteoclasts and osteocytes. After ten cycles, although signal was increased in osteoclasts and osteocytes, it appeared to be decreased in osteoblasts, suggesting that overamplification leads to loss of target complex from these cells. We conclude that several cell types in human bone express ER mRNA in vivo.

Bone Marrow↗
PubMed1997

Quantification of hexokinase mRNA in mouse blastocysts by competitive reverse transcriptase polymerase chain reaction.

Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription-nested polymerase chain reaction (RT-nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT-nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95 x 10(-18) g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation.

Animals↗
PubMed1996

Preoperative diagnosis of thyroid papillary carcinoma by reverse transcriptase polymerase chain reaction of the MUC1 gene.

As thyroid nodules are common, it is imperative to recommend operation only to those with a high risk of malignancy. Fine-needle aspiration (FNA) biopsy, which is widely used for this purpose, is limited by the considerable rate of non-diagnostic or non-interpretable conclusions. Therefore, it is highly desirable to acquire a new diagnostic means for thyroid cancer. We have recently described a marked over-expression of the MUC1 gene in thyroid papillary-carcinoma tissue, as compared with various benign thyroid pathologies. As the amount of mRNA obtainable by FNA is not amenable to hybridization analysis, we amplified the mRNA sequence of the MUC1-gene upstream of the variable number tandem repeat array using the reverse-transcription polymerase chain reaction (RT-PCR). Seven out of 8 FNA samples obtained from thyroid papillary carcinoma resulted in 336 and 309 base-pair products. In contrast, in all 13 FNA samples obtained from various benign pathologies, only the smaller RT-PCR product was observed. Sequence analysis of the RT-PCR products indicates that alternative splicing of the exon 2 acceptor site accounts for the difference between the 2 amplification products. It is suggested that RT-PCR of the MUC1-gene transcript may add a biomolecular diagnostic dimension to the routine cytological preoperative FNA diagnosis.

Adult↗
PubMed1995

Competitive reverse-transcriptase polymerase chain reaction without an artificial internal standard.

Advances in our understanding of molecular and cellular physiology necessitate that mRNA levels for specific growth factors and other rare transcripts be measured quantitatively in small samples. Conventional methods such as Northern blot analysis and solution hybridization/ribonuclease protection are not sufficiently sensitive. We now report the theory, development, and validation of a rapid and highly sensitive assay, the RNA/DNA quantitative polymerase chain reaction (RD-PCR), which uses a competitive PCR approach to measure the number of copies of a specific mRNA per cell. Total nucleic acid (RNA and genomic DNA) is isolated from cells in culture. The mRNA of interest is first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3'-end, and a sequence complementary to an intron of the desired gene at the 5'-end. Competitive PCR is then performed in the presence of the cDNA product and endogenous genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest, and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The cell's own genomic DNA is thereby used as the internal standard. To control for the efficiency of reverse transcription, a standard curve is used in each assay. The technique was validated by comparing the quantitation of insulin-like growth factor I (IGF-I) mRNA in two human cell lines by RD-PCR and by RNase protection analysis. Both methods gave similar numbers of copies of IGF-I mRNA per cell. For accurate analysis, RNase protection required at least 10(7) cells; RD-PCR required as little as 10(2) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1998

The role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay.

In May 1994 an outbreak of vomiting and diarrhoea occurred in a 28-bed long-stay ward for the mentally infirm. The predominant symptoms were vomiting, diarrhoea, malaise and abdominal pain lasting for approximately 12 h in most cases. The attack rate was 62% (13/21) for patients and 46% (16/35) for staff members. Infection control measures were implemented (containment of infectious individuals, hand hygiene among staff and environmental decontamination) and the ward was closed to admissions. Affected staff were excluded from contact with patients and their food until asymptomatic for 72 h. The outbreak lasted for 17 days. Faecal samples from nine symptomatic persons were negative for bacterial enteric pathogens, Giardia, Cryptosporidium and group A rotavirus. Electron microscopy of 12 faecal samples and one sample of vomitus revealed small round structured virus (SRSV) particles in one faecal sample. A further 30 faecal samples and seven vomitus samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for SRSV of which 12 (40%) and 1 (14%) were positive respectively. Twenty-eight throat swabs from symptomatic and asymptomatic patients were collected, three (9.5%) of which were positive for SRSV by RT-PCR. Thirty-six environmental swabs were collected on the affected ward, and 11 (30%) were positive by RT-PCR. Positive swabs were from lockers, curtains and commodes and confined to the immediate environment of symptomatic patients. The distribution of contamination supports the rationale of cohorting sick patients.

Cross Infection↗