Fluorescence qRT-PCR Research Articles

MiR-328 inhibits cell apoptosis and improves cardiac function in rats with myocardial ischemia-reperfusion injury through MEK-ERK signaling pathway.

To explore the influences of micro ribonucleic acid (miR)-328 on rats with myocardial ischemia-reperfusion (IR) injury through the methyl ethyl ketone (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway. A total of 36 Sprague-Dawley rats were randomly assigned into the sham group (n=12), model group (n=12), and miR-328 group (n=12). The model of myocardial IR injury was established by ligating the left anterior descending coronary artery, without any intervention in the model group, while 200 μL of miR-328 antagomir was intravenously injected before modeling in the miR-328 group. The activity of the serum myocardial enzymes lactate dehydrogenase (LDH) and creatine kinase-muscle/brain (CK-MB) was determined via ELISA to assess the cardiac function in the three groups of rats, and the mRNA expression level of miR-328 in myocardial tissues was measured through real-time fluorescence qRT-PCR in the sham group, model group, and miR-328 group. TUNEL staining was performed to detect apoptotic cells, and the levels of myocardial apoptosis-associated protein Caspase-3 and phosphorylated MEK1/2 (p-MEK1/2) and p-ERK1/2 proteins were determined using Western blotting. Compared with the sham group, the model group exhibited increased activity of LDH and CK-MB, miR-328 expression level, apoptotic cells, the relative expression level of Caspase-3, and protein levels of p-MEK and p-ERK, with statistically significant differences (p<0.05). Besides, in comparison with the model group, miR-328 group showed a decreased activity of LDH and CK-MB, miR-328 expression level, the relative expression level of Caspase-3, and protein levels of p-MEK and p-ERK, displaying statistically significant differences (p<0.05). MiR-328 modulates the MEK-ERK signaling pathway to inhibit cell apoptosis and improve the cardiac function in rats with myocardial IR injury.

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay

BackgroundFusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management.MethodsWe performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization.ResultsGene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL).ConclusionsWe describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.