TaqMan Reverse Transcriptase-polymerase Chain Research Articles

Differential Expression of LMNA/C and Insulin Receptor Transcript Variants in Peripheral Blood Mononuclear Cells of Leukemia Patients.

Background: Recent research has identified alternative transcript variants of LMNA/C (LMNA, LMNC, LMNAΔ10, and LMNAΔ50) and insulin receptors (INSRs) as potential biomarkers for various types of cancer. The objective of this study was to assess the expression of LMNA/C and INSR transcript variants in peripheral blood mononuclear cells (PBMCs) of leukemia patients to investigate their potential as diagnostic biomarkers. Methods: Quantitative TaqMan reverse transcriptase polymerase chain reaction (RT-qPCR) was utilized to quantify the mRNA levels of LMNA/C (LMNA, LMNC, LMNAΔ10, and LMNAΔ50) as well as INSR (IR-A and IR-B) variants in PBMCs obtained from healthy individuals (n = 32) and patients diagnosed with primary leukemias (acute myeloid leukemia (AML): n = 17; acute lymphoblastic leukemia (ALL): n = 8; chronic myeloid leukemia (CML): n = 5; and chronic lymphocytic leukemia (CLL): n = 15). Results: Only LMNA and LMNC transcripts were notably present in PBMCs. Both exhibited significantly decreased expression levels in leukemia patients compared to the healthy control group. Particularly, the LMNC:LMNA ratio was notably higher in AML patients. Interestingly, IR-B expression was not detectable in any of the PBMC samples, precluding the calculation of the IR-A:IR-B ratio as a diagnostic marker. Despite reduced expression across all types of leukemia, IR-A levels remained detectable, indicating its potential involvement in disease progression. Conclusions: This study highlights the distinct expression patterns of LMNA/C and INSR transcript variants in PBMCs of leukemia patients. The LMNC:LMNA ratio shows promise as a potential diagnostic indicator for AML, while further research is necessary to understand the role of IR-A in leukemia pathogenesis and its potential as a therapeutic target.

Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury

Hantaviruses, one of the causative agents of viral hemorrhagic fevers, represent a considerable healthcare threat. In Hungary, Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) are the main circulating hantavirus species, responsible for the clinical picture known as hemorrhagic fever with renal syndrome, a disease that may be accompanied by acute kidney injury (AKI), requiring hospitalization with occasionally prolonged recovery phase. A total of 20 patient sera were collected over a 2-year period from persons hospitalized with AKI, displaying clinical signs and laboratory findings directly suggestive for hantavirus infection. Samples were tested using an immunoblot assay, based on complete viral nucleocapsid proteins to detect patients' IgM and IgG antibodies against DOBV and PUUV. In parallel, all specimens were also tested by 1-step real-time TaqMan reverse-transcriptase polymerase chain reaction to confirm infection and to determine the causative hantavirus genotype. We present here the first Hungarian clinical study spanning across 2 years and dedicated specifically to assess acute kidney injuries, in the context of hantavirus prevalence.

Quantification of “bacterial load” in late-onset neonatal sepsis using molecular techniques

Premature infants are at increased risk of bacterial sepsis. Few studies of neonatal infection have quantified the degree of “bacterial load”. Traditionally, bacterial numbers have been estimated by measuring colony-forming-units per ml (CFU/ml), using conventional culture methods. Modern molecular techniques allow direct estimation of bacterial numbers in preterm infants with late-onset sepsis. Levels of bacterial 16S ribosomal RNA (rRNA) can potentially be used to quantify metabolically active bacteria in clinical samples. Objective To estimate the “bacterial load” in infants presenting with late-onset bacterial sepsis (>48 h old) using quantitative molecular methods. Methods A single-centred, observational study was conducted in all infants greater than 48 h old with clinically suspected sepsis. Blood samples (0.5 ml) were taken for a calibrated TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) in addition to normal infection investigations. This assay allows quantitative detection of bacterial 16S rRNA and rDNA. Clinical and laboratory data were recorded at the time of sampling. Copy numbers (rRNA and rDNA) were calculated and corrected for background genetic material to estimate bacterial load. Results 100 samples were taken from 125 babies at a median age of 14 days. Median (interquartile) birth weight was 1.09 (0.81–1.45) kg and gestation was 27.5 (23–40) weeks. The rate of clinically diagnosed late-onset infection was 33%. The mean calculated and corrected copy number estimate (bacterial rRNA+rDNA) was 1.51×107/ml of blood (range 8.91×104 to 9.77×1010). Estimated copy number correlated positively with white blood cell count, neutrophil number and C-reactive protein and negatively with platelet count, lymphocyte count, pH and base deficit. There was no correlation with birth weight or gestational age. Conclusion Bacterial load in late-onset sepsis can be estimated by quantitative RT-PCR using a 16S rRNA assay. Direct comparison to estimates of CFU/ml was not possible because of the variability of rRNA numbers per bacterial cell. However, the potential of this target as a quantitative biomarker of bacterial infection was demonstrated. Increasing numbers of bacteria appear to stimulate greater immune response. Further study is necessary to place these values in context with other age groups and disease severity.

RET/PTC Rearrangement Occurring in Primary Peritoneal Carcinoma

RET/PTC rearrangements are initiating events in the development of a significant proportion of papillary thyroid carcinomas. Activated RET/PTC mutations are thought to be restricted to thyroid disease, but this study proposes that these events may also occur in nonthyroid tumors. A total of 57 nonthyroid papillary tumors were examined for RET/PTC rearrangements using interphase fluorescence in situ hybridization, Taqman reverse transcriptase polymerase chain reaction, and immunohistochemistry. Taqman single nucleotide polymorphism detection was used to analyze for expression of mutated BRAF T1799A. In all, 20% (3/15) of primary peritoneal carcinoma had detectable RET/PTC1 rearrangements by all 3 methodologies. A further case of similar histotype had an alternate RET/ PTC rearrangement. No RET/PTC1 rearrangements were detected in the remaining tumor cohort. All 57 tumors were homozygous for wild-type BRAF. The results indicate that RET/PTC rearrangements occur in a small subset of nonthyroid papillary tumors. These rearrangements may not be directly implicated in tumor growth; rather representing "passenger" mutations reflecting RET instability in secondary tumor subclones.

Differential gene expression of somatotrophic and growth factors in response to in vivo hypoxia in human placenta

Hypoxia-inducible transcription factors (HIFs) have been characterized as the most important regulators of O(2)-dependent gene transcription. We investigated expression of HIF-dependent growth factors and HIF-independent somatotrophic factors in term placenta in response to hypoxic ischemia. Our cross-sectional in vivo analysis included term placentas of gestations complicated by the following: (1) birth asphyxia (n = 22); (2) chronic hypoxic ischemia (n = 22); and (3) controls (n = 28). Gene expression of leptin, insulin-like growth factor (IGF)-1, IGF-2, ghrelin, and human placental growth hormone (hPGH) were measured by TaqMan reverse transcriptase-polymerase chain reaction. Acute and chronic hypoxia significantly increased leptin messenger ribonucleic acid (mRNA) levels, compared with controls (P < .001). Augmented IGF-2 mRNA levels were present in chronic hypoxia (P < .001) but not in birth asphyxia. IGF-1, ghrelin, and hPGH mRNA levels did not change in relation to hypoxia. IGF-2 and leptin are suggested to be involved in adaptive response to hypoxic ischemia in term placenta with differential transcriptional regulation related to the duration of hypoxia.

CLINICAL STUDY: Endogenous morphine signaling via nitric oxide regulates the expression of CYP2D6 and COMT: autocrine/paracrine feedback inhibition

We determined changes in mRNA expression in specific enzymes involved in the biosynthesis of morphine in human white blood cells via microarray. Leukocyte exposure to morphine down-regulated catechol-O-methyl transferase (COMT) and CYP2D6 by approximately 50% compared with control values. The treatment did not alter DOPA decarboxylase and dopamine beta-hydroxylase expression, demonstrating the specificity of morphine actions. The verification of the microarray data was accomplished via real-time Taqman reverse transcriptase polymerase chain reaction (RT-PCR) focused on CYP2D6 and COMT expression in different blood samples treated with morphine. The analysis showed similar changes in the expression of CYP2D6 and COMT mRNA. The expression was reduced by 47 +/- 7% for CYP2D6, substantiating the microarray finding of a 54% reduction. Furthermore, exposure of white blood cells to 10(-6) M S-nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, reduced the expression of CYP2D6 and COMT. Prior naloxone (10(-6) M) or N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) addition abrogated morphine's down-regulating activity, demonstrating morphine was initiating its actions via stimulating constitutive NO synthase derived NO release via the mu3 opiate receptor splice variant. In the past we demonstrated that UDP-glucurosyltransferase is involved in metabolizing morphine to morphine 6-glucuronide in adrenal chromaffin cells. In the present study its expression was not found in controls and morphine-treated cells, suggesting that morphine 6-glucuronide may not be synthesized in white blood cells. Taken together, it appears that morphine has the ability to modulate its own synthesis via autocrine and paracrine signaling.

Rapid Detection of Highly Pathogenic Avian Influenza H5N1 Virus by TaqMan Reverse Transcriptase–Polymerase Chain Reaction

Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.

Optimizing Aerosol Gene Delivery and Expression in the Ovine Lung

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

A Survey for West Nile Virus in Bats from Illinois

A blocking enzyme-linked immunosorbent assay was used to test 97 serum samples from big brown bats (Eptesicus fuscus) captured in six counties in Illinois between May 2002 and February 2004 for West Nile virus (WNV) antibodies. One female big brown bat tested positive for WNV antibodies. Samples of kidney, liver, and heart tissue were collected from 312 bats of seven species that were submitted to the Illinois (USA) Department of Public Health or the Illinois Department of Agriculture diagnostic laboratories between January 2001 and December 2003. Tissue samples were tested for WNV using TaqMan reverse transcriptase polymerase chain reaction and all were negative. Prevalence of WNV antibodies in the bats (1%) was lower than previously reported for other flaviviruses, but similar to the prevalence (2%) of WNV antibodies reported in bats from New Jersey and New York, USA. Additional research is needed to determine potential impact of WNV infections on bats and to determine whether they play a role in the WNV transmission cycle.

A COMPARISON OF TWO WEST NILE VIRUS DETECTION ASSAYS (TAQMAN REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION AND VECTEST ANTIGEN ASSAY) DURING THREE CONSECUTIVE OUTBREAKS IN NORTHERN ILLINOIS

Mosquitoes identified as female Culex (Culex) species, primarily mixtures or uniform batches of Culex pipiens and Culex restuans, were collected daily from gravid traps by 2 mosquito abatement districts (MADs) in Cook County, Illinois. From 2002 through 2004, batches (pools) of mosquitoes were tested by the MADs for West Nile virus (WNV) by using VecTest WNV antigen assays and the same samples were retested, usually within 1-2 wk, for WNV RNA by the TaqMan reverse transcriptase polymerase chain reaction (RT-PCR). There were 952 TaqMan-positive pools out of 3,953 pools over the 3 years, and about one half of that number were VecTest-positive. The difference between the 2 detection assays varied between and within years. The VecTest assays detected about 57% and 69% of the TaqMan RT-PCR-positive pools from Des Plaines Valley MAD and Northwest MAD in 2002, but only about 40% and 46% in 2003, and 36% and 55% in 2004, respectively. Based on a subset of the 2004 data, a linear relationship was found between VecTest detection of WNV and TaqMan cycle threshold between 18 and 28 cycles. A temporal decrease in the difference between the 2 assays was observed in 2003 and 2004, which we conjecture is due, at least partially, to a seasonal decline in the proportion of recently infected mosquitoes. This trend was not observed in 2002 because infection rates indicated a high likelihood of more than 1 infected mosquito per pool at the peak of transmission. Unlike a previous study, the 95% confidence intervals of infection rates based on the 2 detection methods did not always overlap. The highest infection rates occurred in 2002 when mean monthly temperatures were above average.

Transcription of the inositol polyphosphate 1-phosphatase gene (INPP1) is upregulated in human colorectal cancer.

We have used suppression subtractive hybridization to demonstrate significant overexpression of the inositol polyphosphate 1-phosphatase gene (INPP1) in colorectal cancer compared with matched normal colon epithelium. Its gene product catalyses the hydrolysis of inositol 1,3,4-trisphosphate and inositol 1, 4-bisphosphate, a key molecule in the phosphoinositide metabolic and signaling pathways. Following confirmation of the differential expression by reverse Northern dot blot analysis, fully quantitative Taqman reverse transcriptase-polymerase chain reaction assays showed that its transcription was upregulated in 42/49 colorectal tumors. There was no significant difference in four tumors and reduced transcription was observed in three. This is the first study to report the upregulation of the INPP1 gene in a human cancer and should facilitate further studies looking at the role of phosphatidylinositol signaling reactions in human colorectal cancer.