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Selection of reference genes for quantitative studies in acute myeloid leukaemia

Lee C, Yiau KXS, Lee LJ, Chong PP, Chang KM, Abdullah M

Malays J Pathol, 2019 Dec;41(3):313-326.
PMID: 31901916

INTRODUCTION: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed.
MATERIALS AND METHODS: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12).
RESULTS: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns.
CONCLUSIONS: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.

Matched MeSH terms: Bone Marrow/metabolism
Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

Mamidi MK, Nathan KG, Singh G, Thrichelvam ST, Mohd Yusof NA, Fakharuzi NA, et al.

J Cell Biochem, 2012 Oct;113(10):3153-64.
PMID: 22615164 DOI: 10.1002/jcb.24193

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo-, chondro-, and adipo-lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy.

Matched MeSH terms: Bone Marrow/metabolism*
Fulltext Autologous serum supplement favours in vitro regenerative paracrine factors synthesis

Haque N, Kasim NHA, Kassim NLA, Rahman MT

Cell Prolif, 2017 Aug;50(4).
PMID: 28682474 DOI: 10.1111/cpr.12354

OBJECTIVES: Foetal bovine serum (FBS) is often the serum supplement of choice for in vitro human cell culture. This study compares the effect of FBS and autologous human serum (AuHS) supplement in human peripheral blood mononuclear cell (PBMC) culture to prepare secretome.
MATERIALS AND METHODS: The PBMC (n = 7) were cultured either in RPMI-1640 containing L-glutamine and 50 units/ml Penicillin-Streptomycin (BM) or in BM with either AuHS or FBS. Viability, proliferation and differentiation of PBMC were evaluated. Paracrine factors present in the secretomes (n = 6) were analysed using ProcartaPlex Human Cytokine panel (17 plex). Ingenuity Pathway Analysis (IPA) was performed to predict activation or inhibition of biological functions related to tissue regeneration.
RESULTS: The viability of PBMC that were cultured with FBS supplement was significantly reduced at 96 h compared to those at 0 and 24 h (P

Matched MeSH terms: Bone Marrow/metabolism
Protective effects of Etlingera elatior extract on lead acetate-induced changes in oxidative biomarkers in bone marrow of rats

Haleagrahara N, Jackie T, Chakravarthi S, Rao M, Pasupathi T

Food Chem Toxicol, 2010 Oct;48(10):2688-94.
PMID: 20600524 DOI: 10.1016/j.fct.2010.06.041

Several environmental toxins with toxic effects to the bone marrow have been identified. Pathology associated with lead intoxication is due to the cellular damage mediated by free radicals. In the current study, we examined the effect of Etlingera elatior extract on lead-induced changes in the oxidative biomarkers and histology of bone marrow of rats. Sprague-Dawley rats were exposed to 500 ppm lead acetate in their drinking water for 14 days. E. elatior extract was treated orally (100mg/kg body weight) in combination with, or after lead acetate treatment. The results showed that there was a significant increase in lipid hydroperoxide, protein carbonyl content and a significant decrease in total antioxidants, super oxide dismutase, glutathione peroxidase and glutathione--S-transferase in bone marrow after lead acetate exposure. Treatment with E. elatior decreased lipid hydroperoxides and protein carbonyl contents and significantly increased total antioxidants and antioxidant enzymes. Treatments with E. elatior extract also reduced, lead-induced histopathological damage in bone marrow. In conclusion, these data suggest that E. elatior has a powerful antioxidant effect, and it protects the lead acetate-induced bone marrow oxidative damage in rats.

Matched MeSH terms: Bone Marrow/metabolism*