scholarly journals Molecular diagnosis of Iraqi chronic myeloid leukemia patients using quantitative real-time PCR

2010 ◽  
Vol 4 (2) ◽  
pp. 64-69
Author(s):  
Adeeb Abbas ◽  
Majeed A. Sabbah ◽  
Abdul-salam Hatam ◽  
Luma A. Yasser ◽  
Baan Abdul-Latif
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Rna Extraction ◽  
Fusion Transcript ◽  
Quantitative Real Time Pcr ◽  
Rt Pcr ◽  
Chronic Granulocytic Leukemia ◽  
Granulocytic Leukemia

hronic myeloid leukemia (CML), also known as chronic granulocytic leukemia, is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulation of these cells in peripheral blood. Total RNA extraction, cDNA and quantitative real-time PCR (Q-RT-PCR) were done for thirty CML Iraqi patients. Hematology tests (hemoglobin, platelets and WBCs counts) were done for the same samples in the same time. The results of this study show that some samples with normal hematology values have BCR-ABL (p210) fusion transcript with molecular analysis by Q-RT-PCR. This indicates the importance of this technique in the diagnosis and monitoring the therapy of CML patients.

Use of Plasma RNA for Real-Time Quantitative RT-PCR to Monitor Imatinib Therapy in Patients with Chronic Myeloid Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1099-1099
Author(s):  
Vilmos A. Thomazy ◽  
Hagop M. Kantarjian ◽  
Maha Imam ◽  
Peter J. Davies ◽  
Christine Aguilar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Imatinib Therapy ◽  
Rt Pcr ◽  
Cytogenetic Remission ◽  
Plasma Rna

Abstract Monitoring minimal residual disease (MRD) in chronic myeloid leukemia after imatinib therapy can help in determining therapeutic strategy. Sensitive methods based on the detection of the BCR/ABL fusion gene or its fusion transcript by FISH or RT-PCR, respectively, are used to monitor residual leukemia in peripheral blood or bone marrow cells. While sensitive, these methods require an invasive procedure to obtain bone marrow. The patchy nature of the residual disease may cause some of the reported variation in detecting MRD. We hypothesized that plasma RNA level reflects total body disease and is unaffected by the tendency of some myeloid cells not to circulate. We measured the BCR/ABL fusion transcript by real-time quantitative RT-PCR using RNA isolated from blood plasma. We used a quantitative RT-PCR assay capable of measuring both the b2a2 and b3a2 BCR/ABL fusion transcripts by targeting exon 13 of BCR with the 5′ and exon 2 of ABL by the 3′ primer. RNA isolated from 10–30 μl of frozen plasma was subjected to real-time quantitative RT-PCR using the Taqman technology. Quantification was achieved by extrapolation from a standard curve generated from a ten-fold dilution series of a synthetic oligonucleotide corresponding to the (+) strand of the target sequence. To normalize the fusion mRNA levels we also measured β-actin mRNA from the same samples. Assays were run in duplicate. The results of plasma RT-PCR were correlated with results of conventional cytogenetic analysis, FISH, and quantitative RT-PCR of bone marrow suspension (“bone marrow PCR”). Plasma and bone marrow samples were obtained at 3, 6, 9, 12 and 15 months after imatinib therapy. Ninety-five plasma samples were analyzed. There was a strong positive correlation between the results of plasma RT-PCR vs. positive cytogenetics (n=95, p<0.00001), FISH (n=88, p<0.00001), and bone marrow RT-PCR (n=74, p<0.00001). At 12 months after imatinib therapy all but 2 of the 29 patients were in complete hematologic and cytogenetic remission (CR); one patient was in partial remission (PR) and one in hematologic, but not cytogenetic, remission. These two non-CR patients were positive by both bone marrow and plasma-based RT-PCR assay for the fusion transcript. Three of the four CR patients positive by both plasma and bone marrow PCR at 12 months progressed and converted to PR/minimal response at 15 months post-therapy. The level of plasma BCR/ABL fusion mRNA at 12 months was significantly higher (p=0.04) in these patients than in those who did not progress. Our results show that, by using 10–30 μl of plasma per assay, RT-PCR is at least as sensitive as cytogenetic or FISH analysis of bone marrow cells in monitoring MRD.

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

scholarly journals A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽  
2020 ◽  
Vol 10 (12) ◽  
Author(s):  
Siti Suriawati Badai ◽  
Omar Abd Rasid ◽  
Ghulam Kadir Ahmad Parveez ◽  
Mat Yunus Abdul Masani
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Oil Palm ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr

scholarly journals DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

2016 ◽  
Vol 3 (1) ◽  
pp. 7 ◽  
Author(s):  
Michael Lebuhn ◽  
Jaqueline Derenkó ◽  
Antje Rademacher ◽  
Susanne Helbig ◽  
Bernhard Munk ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Interlaboratory Comparison ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Dna And Rna

scholarly journals Detection of the BCR-ABL Gene By the Real-Time PCR Method in Patients with Chronic Myeloid Leukemia in Rio Grande Do Norte, Brazil

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5432-5432
Author(s):  
Aldair Sousa Paiva ◽  
Hugo Diogenes De Oliveira Paiva ◽  
Geraldo Barroso Cavalcanti ◽  
Gioconda DR Leão ◽  
Marcos Dias Leão ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Rna Extraction ◽  
Philadelphia Chromosome ◽  
Genetic Material ◽  
Chromosome 9 ◽  
The Real

Abstract Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients. Disclosures No relevant conflicts of interest to declare.

Detection of Cryptosporidium parvum and Giardia lamblia in Water Supply by Quantitative Real-Time PCR

2012 ◽  
Vol 518-523 ◽  
pp. 3707-3711
Author(s):  
Peng Peng ◽  
Rui Bao Jia ◽  
Yu Mei Liu ◽  
Li Li
Keyword(s):  
Water Supply ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Giardia Lamblia ◽  
Pcr Primers ◽  
Quantitative Real Time Pcr ◽  
Rt Pcr ◽  
High Recovery ◽  
Pcr Method

Cryptosporidium parvum and Giardia lamblia are common pathogenic protozoa in water, which pose high risk to drinking water supply. In the present study, detection of C. parvum and G. lamblia was performed by quantitative real-time PCR (RT-PCR). Pairs of PCR primers were evaluated for the detection specificity to pathogenic C. parvum and G. lamblia. The recovery of the RT-PCR detection procedure was examined and high recovery rates (i.e., more than 45％ for C. parvum and more than 50％ for G. lamblia ) were achieved. The RT-PCR method was used to detect C. parvum and G. lamblia in a secondary water supply. The results indicated the potential application of the quantitative RT- PCR method in detection of C. parvum and G. lamblia in water supply.

Leukemia-Related Gene Expression of Bone Marrow Cells from Patients with Shwachman-Diamond Syndrome at the Pre-Leukemic Phase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3433-3433
Author(s):  
Piya Rujkijyanont ◽  
Joseph Beyene ◽  
Kuiru Wei ◽  
Yigal Dror
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Real Time ◽  
Differential Expression ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
P Values ◽  
Shwachman Diamond Syndrome ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract Background: Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder characterized by varying degrees of cytopenia and a high propensity for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in up to 36% of the patients by the age of 30 years. Although the gene associated with SDS, SBDS, has recently been identified, its function, the link with MDS/AML and the mechanism for the development of MDS/AML in SDS is unclear and the molecular events occurring during transformation haven’t been yet identified. It is likely that several events occur many years before overt transformation occurs, and might be identifiable by comprehensive analysis. Objectives: To use oligonucleotide microarray to identify leukemogenic gene expression before overt transformation, which can explain a propensity for MDS/AML. Methods: Total RNA from marrow cells from 9 SDS patients and 7 healthy age-matched donors of bone marrows for transplantation was extracted, labeled and hybridized to Affymetrix HG_U133_Plus2.0 GeneChip. Data were pre-processed using robust multichip analysis (RMA) and differentially expressed genes were identified with permutation-based methods. False discovery rate (FDR)-adjusted p-values were used to rank genes and cluster analysis grouped genes and samples. Real-time PCR was performed to confirm differential expression of genes found by microarray. Results: Of the 38,500 genes on the HG_133_Plus2.0 we analyzed 52 known leukemia-related genes. We identified several genes with small FDR-adjusted p-values. Clustering of arrays resulted in two clusters that clearly separated patients from controls. Interestingly among the leukemia-related genes, the most differentially expressed gene (T=4.2) was ARHGEF12, a member of the Rho GEF family. Rho GEFs are oncogenes; many of them can transform NIH 3T3 cells into a malignant phenotype by altering expression and activation of Rho GTPases. ARHGEF12 is mapped at 11q23, telomeric to MLL, and is a novel MLL fusion partner in acute myeloid leukemia. Real time PCR after normalization against beta-actin confirmed statistically higher expression of the ARHGEF12 (p=0.03) in SDS marrow cells. In addition to ARHGEF12, we have found striking expression changes in several other genes, related to MDS/AML including TAL1, whose differential expression was also confirmed by real-time PCR. Conclusions: SDS marrow cells exhibit abnormal gene expression pattern, which might results in continuous stimulation favoring evolution or progression of malignant clones. Additional molecular and cytogenetic events are likely necessary for the malignant process to be irreversible and complete. Although analysis of whole marrow cells may not enable the detection of genes with lower differential expression between SDS and normal, it may still assist identifying molecular pathways involved in leukemogenesis. This is critically important when studying marrow failure disorders as obtaining sufficient amount of RNA from purified cell population is largely impossible.

Sign in / Sign up

Export Citation Format

Share Document