ABSTRACT
Endogenous housekeeping genes are traditionally employed to normalize the expression of target genes in RT-qPCR studies. Assuming that a perfect housekeeping suitable for every condition does not exist, expression stability of the chosen reference gene should be evaluated at every new experiment. The housekeeping selection process reveals furthermore complicated and time-consuming when different conditions have to be compared in the same experimental dataset. As an alternative strategy, we spiked an external reference transcript (ERT) into all RNA samples of our dataset (eggplant roots subjected to different biotic stresses), and used it to normalize the expression levels of native candidate housekeeping. ERT expression resulted highly stable across all samples and enabled to indicate glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the most stable endogenous housekeeping. This result was confirmed by the use of GeNorm, Normfinder, and BestKeeper algorithms. This method might be generally applied to expedite the selection process of the best reference gene.
Subject(s)
Gene Expression Profiling/standards , Plant Roots/genetics , Solanum melongena/genetics , Gene Expression Regulation, Plant , Genes, Essential , Genes, Plant , Plant Diseases/microbiology , Plant Roots/metabolism , Plant Roots/microbiology , Real-Time Polymerase Chain Reaction , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction , Solanum melongena/metabolism , Solanum melongena/microbiology , Stress, PhysiologicalABSTRACT
Recently, Dawson et al identified a previously unrecognized nuclear role of JAK2 in the phosphorylation of histone H3 in hematopoietic cell lines. We searched nuclear JAK2 in total bone marrow (BM) cells and in 4 sorted BM cell populations (CD34(+), CD15(+), CD41(+), and CD71(+)) of 10 myeloproliferative neoplasia (MPN) patients with JAK2V617F mutation and 5 patients with wild-type JAK2 MPN. Confocal immunofluorescent images and Western blot analyses of nuclear and cytoplasmic fractions found nuclear JAK2 in CD34(+) cells of 10 of 10 JAK2-mutated patients but not in patients with wild-type JAK2. JAK2 was predominantly in the cytoplasmic fraction of differentiated granulocytic, megakaryocytic, or erythroid cells obtained from all patients. JAK2V617F up-regulates LMO2 in K562 and in JAK2V617F-positive CD34(+) cells. The selective JAK2 inhibitor AG490 normalizes the LMO2 levels in V617F-positive K562 and restores the cyto-plasmic localization of JAK2.
Subject(s)
Antigens, CD34/metabolism , Cell Nucleus/metabolism , Erythroid Cells/metabolism , Granulocytes/metabolism , Janus Kinase 2/genetics , Megakaryocytes/metabolism , Myeloproliferative Disorders/metabolism , Philadelphia Chromosome , Apoptosis , Blotting, Western , Bone Marrow/metabolism , Cells, Cultured , Cytoplasm/metabolism , Granulocytes/cytology , Humans , Janus Kinase 2/metabolism , K562 Cells , Megakaryocytes/cytology , Microscopy, Fluorescence , Mutation/genetics , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Phosphorylation , Prognosis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionSubject(s)
Cell Transformation, Neoplastic/genetics , Janus Kinase 2/genetics , Leukemia, Myeloid, Acute/genetics , Mutation , Myeloproliferative Disorders/genetics , Adult , Aged , Aged, 80 and over , Bone Marrow Cells/pathology , Cohort Studies , Europe , Female , Flow Cytometry , Granulocyte Precursor Cells/pathology , Humans , Male , Middle AgedABSTRACT
The kneeling view is a method to objectively measure posterior knee laxity. However, the actual amount of load applied and the reliability of this method in term of side to side difference are not known. We studies these issues in a group a 25 healthy volunteers who underwent measurements of posterior knee laxity in both knees. A standard digital scale was positioned under both kneeling supports to measure the actual amounts of posterior displacement forces applied. We measured the mass of the subject, the side-to-side difference of the weight applied into anterior aspect of the tibia, and the ratio weight of the subject/ amount of posterior displacement load applied. The average amount of forces applied was at least 75% of the body weight of each subject, with a side-to-side variability of 3.3% of the weight applied. The kneeling view can be considered, in terms of forces applied, a reliable and reproducible alternative method for the routine radiographic evaluation of the posterior knee laxity.
Subject(s)
Biomechanical Phenomena , Joint Instability/diagnosis , Knee Joint/physiology , Physical Examination/methods , Adult , Female , Humans , Male , Reference Values , Young AdultABSTRACT
A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.
