Objective
This study intended to establish a droplet digital PCR (dd‐PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)‐positive chronic myeloid leukemia (CML), ...thereby achieving deep‐level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment.
Methods
Using dd‐PCR and RT‐qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML‐chronic phase (CML‐CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow‐ups. By RT‐qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd‐PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods.
Results
At the cellular level, consistency of results of dd‐PCR and RT‐qPCR reached R2 ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd‐PCR results; X: RT‐qPCR results). In the dd‐PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow‐up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd‐PCR 3 months earlier than by RT‐qPCR.
Conclusion
In contrast with RT‐qPCR, dd‐PCR is more sensitive, thus enabling accurate conversion of dd‐PCR results into internationally standard RT‐qPCR results by conversion equation, to achieve a deeper molecular biology‐based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.
Developing a simple and cost-effective strategy to diagnose and treat cancer with a single and minimum dose through noninvasive strategies is highly challenging. Nano-sized graphene and graphene ...oxide (GO) are promising for biomedical applications, such as drug delivery and the photo thermal therapy of cancer. An acute promyelocytic leukemia line, such as NB4 cells, was detected by modified GO in extracellular and intracellular experiments. Results shows that GO can quench the fluorescence of the ssDNA fluorescent probe and its fluorescence is restored after a PML/RARα fusion gene of NB4 cell lines is added. Therefore, the fusion gene can be detected accurately with this phenomenon. The best detection conditions for the fusion gene are found with assDNA fluorescent probe concentration of 200 nmol/L in the presence of 0.04 mg/L GO at room temperature for 1 h.
Identification of a novel circularized transcript of the AML1 gene Xu, A.Nn., The Second Hospital of Shanxi Medical University, Taiyuan, China; Chen, X.H., The Second Hospital of Shanxi Medical University, Taiyuan, China; Tan, Y.H., The Second Hospital of Shanxi Medical University, Taiyuan, China ...
BMB Reports,
03/2013, Volume:
46, Issue:
3
Journal Article
Peer reviewed
Open access
The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs ...are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outwardfacing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.
The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs ...are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outwardfacing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.
This study was purposed to investigate the relation of CD25 with the acute B cell lymphoblastic leukemia (B-ALL) and its clinical significance. A totol of 88 newly diagnosed B-ALL patients were ...enrolled in this study. The immunophenotype of leukemic myeloblasts were detected by flow cytometry, including interleukin 2 receptor α chain (CD25), β chain (CD122), γ chain (CD132), CD19, CD20, CD10, CD34, CDIgM, CD79a, CD22 and CDTDT. The expression of BCR/ABL fusion gene was detected by qualitative PCR. The expression of IL2RA (CD25 gene) was detected by real-time qualitative RT-PCR. The results showed that there was no significant statistical difference in WBC count, Hb level, PLT count, marrow blast rate, peripheral blast rate, hepato-lienal infiltration, lymph node infiltration, levels of CD10, CD20, CD22, CD34, CD79a, CDTDT, CDIgM expression between B-ALL patients with CD25(+) and B-ALL patients with CD25(-), while the CD19 expression level in B-ALL patients with CD25(+) was higher than that in B-ALL patients w
To explore the effect of nuclear factor erythroid-2 related factor 2 (Nrf2) and thioredoxin reductase (TrxR) gene on proliferation of chronic myeloid leukemia (CML) line cells and its mechanism.
Four ...interfering sequences of Nrf2 and one negative control sequence were designed and synthesised based on the principle of target sequence of siRNA, then constructed lentivirus vectors, which were transfected into K562 cell lines. The transfection effect was observed by laser scanning confocal microscope (LSCM) and flow cytometer (FCM); The depressing effect of siRNA was analyzed by real-time PCR. The cell proliferation inhibiting rate was measured with CCK-8 assay, the apoptotic rate by Annexin V-PE/PI with FCM and the apoptotic morphology of cells by LSCM.
The transfection efficiency of lentivirus was 65%. One cell line K562-C3 which significantly inhibited Nrf2 mRNA was obtained by real-time PCR, Nrf2 relative quantitation (RQ) expressions were 1.003±0.093 and 0.344±0.032 in the control group and K562-C3 respecti
The aim of this study was to detect the rate of T-helper (Th)17 cells and interleukin (IL)-17 level in peripheral blood of patients with primary immune thrombocytopenia (ITP) and to explore their ...clinical significance. The proportion of Th17 cells from 48 patients with ITP and 28 healthy controls was detected by flow cytometry, and the IL-17 level was evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that the percentage of Th17 cells in ITP group was (1.40 ± 1.35)%, which was significantly higher than that in healthy control group (P < 0.05), but in the glucocorticoid hormone-treated group it was significantly lower than that in treated group without glucocorticoid hormone(P < 0.05). The level of IL-17 expressed by Th17 cells in ITP patients was (19.624 ± 5.187) pg/ml, which was higher than that in the healthy control group (P < 0.05), it was lower in the glucocorticoid hormone treated group than that in treated group without glucocorticoid hormone, but there was no statistically sign
To screen the potential protein biomarkers in minimal residual disease (MRD) of the acute promyelocytic leukemia (APL) by comparison of differentially expressed serum protein between APL patients at ...diagnosis and after complete remission (CR) and healthy controls, and to establish and verify a diagnostic model.
Serum proteins from 36 cases of primary APL, 29 cases of APL during complete remission and 32 healthy controls were purified by magnetic beads and then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The spectra were analyzed statistically using FlexAnalysis(TM) and ClinProt(TM) software.
Two prediction model of primary APL/healthy control, primary APL/APL CR were developed. Thirty four statistically significant peptide peaks were obtained with the m/z value ranging from 1000 to 10 000 (P < 0.001) in primary APL/healthy control model. Seven statistically significant peptide peaks were obtained in primary APL/APL CR model (P < 0.001). Comparison o
Developing a simple and cost-effective strategy to diagnose and treat cancer with a single and minimum dose through noninvasive strategies is highly challenging. Nano-sized graphene and graphene ...oxide (GO) are promising for biomedical applications, such as drug delivery and the photo thermal therapy of cancer. An acute promyelocytic leukemia line, such as NB4 cells, was detected by modified GO in extracellular and intracellular experiments. Results shows that GO can quench the fluorescence of the ssDNA fluorescent probe and its fluorescence is restored after a PML/RARα fusion gene of NB4 cell lines is added. Therefore, the fusion gene can be detected accurately with this phenomenon. The best detection conditions for the fusion gene are found with assDNA fluorescent probe concentration of 200nmol/L in the presence of 0.04mg/L GO at room temperature for 1h.
New Carbon Materials 2014, 29(6): 438–443
PDF
