Real-Time Quantitative-PCR (qrt-PCR) Using Alleleseqr Technology: to Assess Chimerism in Pediatrics Patients Undergoing Hematopoietic Stem Cell Transplantation (HSCT)

Acute graft-versus-host disease (aGVHD) remains a significant and severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Due to the occurrence of aGVHD, allo-HSCT significantly increases the mortality rate compared with autologous hematopoietic stem cell transplantation (auto-HSCT). In this study, auto-HSCT and allo-HSCT aGVHD mouse models were built to detect the difference in CD4+ lymphocyte in different tissues based on ribonucleic acid sequencing (RNA-Seq) analysis. Clustering analysis, functional annotation, and pathway enrichment analysis were performed on differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was used to find hub genes. CD4+T cells were activated by MLR and cytokine stimulation. Cells were sorted out by a flow cell sorter. The selected genes were verified by qRT-PCR, histology, and immunofluorescence staining. The GSE126518 GEO dataset was used to verify the hub genes. Enrichment analysis revealed four immune-related pathways that play an important role in aGVHD, including immunoregulatory interactions between a lymphoid and a nonlymphoid cell, chemokine receptors binding chemokines, cytokine and cytokine receptor interaction, and the chemokine signaling pathway. At the same time, with the PPI network, 11 novel hub genes that were most likely to participate in immunoregulation in aGVHD were identified, which were further validated by qRT-PCR and the GSE126518 dataset. Besides, the protein expression level of Cxcl7 was consistent with the sequencing results. In summary, this study revealed that immunoregulation-related DEGs and pathways played a vital role in the onset of aGVHD. These findings may provide some new clues for probing the pathogenesis and treatment of aGVHD.

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Chimerism Monitoring after Allogeneic Hematopoietic Stem Cell Transplantation Using Quantitative Real-Time PCR of Biallelic Insertion/Deletion Polymorphisms

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2014.06.005 ◽

2014 ◽

Vol 16 (6) ◽

pp. 679-688 ◽

Cited By ~ 20

Author(s):

Seon Young Kim ◽

Moon Hwan Jeong ◽

Nare Park ◽

Eunkyoung Ra ◽

Hyunwoong Park ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Real Time ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Hematopoietic Stem

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Monitoring JAK3 mRNA Levels in Patients with GVHD after Allogeneic Hematopoietic Stem Cell Transplantation by Real-Time Quantitative PCR.

Blood ◽

10.1182/blood.v108.11.5273.5273 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5273-5273

Author(s):

Qihui Li ◽

Xin Du ◽

Suxia Geng ◽

Jianyu Weng ◽

Rong Guo ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Quantitative Pcr ◽

Conditioning Regimen ◽

Myeloablative Conditioning ◽

Hematopoietic Stem ◽

Real Time Quantitative Pcr

Abstract Background: Graft-versus-host disease (GVHD) represents a major hurdle impeding the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT). JAK3 plays pivotal roles in the initiation of cytokine-triggered signaling transduction. Recent studies about the murine GVHD model show that targeting JAK3 in donor lymphocytes with a chemical inhibitor such as WHI-P131 may be useful in the prevention of severe GVHD. However, the level of JAK3 expression in patients with GVHD has not been reported. The current study is to develop a reliable and rapid real-time quantitative PCR (Q-PCR) method using Taqman Probe for detecting JAK3 in allo-HSCT recipients. Methods: 20 patients who received myeloablative-conditioning regimen for hematological malignancies and cyclosporine for GVHD prophylaxis have been investigated in this still ongoing study clinically. One of these patients developed acute GVHD (aGVHD), 9 patients developed chronic GVHD (cGVHD), and 10 patients showed no evidence of GVHD during the first 365 days post-transplant. RNA was extracted from white blood cells and cDNA templates were synthesized using 1μg RNA. The standard curve method was used for relative quantitation of JAK3. Results: There is a clear trend for a increasing expression of JAK3 in 9 patients with GVHD. Interestingly, a significant decrease of JAK3 gene expression when the severity of GVHD was remission (P=0.04). One of the patient with cGVHD, the level of JAK3 gene expression has no changed before and after treatment. However, these patients post HSCT without GVHD have a comparable level of JAK3 expression before myeloablative conditioning regimen(P>0.05). Conclusion: Based on our preliminary results, JAK3 mRNA level was obviously correlated with the development of GVHD. Analysis JAK3 mRNA expression may be useful in evaluating GVHD patients with allogeneic stem cell transplantation.

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Quantitative PCR technology in chimerism status evaluation after hematopoietic stem cell transplantation

Molecular and experimental biology in medicine ◽

10.33602/mebm.2.1.8 ◽

2019 ◽

Vol 2 (1) ◽

pp. 44-47

Author(s):

Katarina Stingl Jankovic

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Quantitative Pcr ◽

Hematopoietic Stem Cell ◽

Disease Relapse ◽

Hematopoietic Stem ◽

Qpcr Method ◽

Pcr Technology

Chimerism status evaluation is one of the most useful methods for the assessment of the hematopoietic stem cell transplantation outcome, since it gives information about engraftment and disease relapse, as well as providing indication for timely interventions such as donor lymphocyte infusions. The aim of this article is to give an overview of the different methods used for chimerism detection, with a especial emphasis on the quantitative PCR (qPCR) based techniques as the newest in line of techniques used for chimerism monitoring. This overview covers a description of the qPCR method as well as a discussion about the advantages this technique offers in comparison to other methods.

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