Preanalytical mRNA Stabilization of Whole Bone Marrow Samples.

Abstract Background: Gene expression profiling is a useful tool for the diagnosis and basic research of cancer. One of the major limitations of this approach is that already a short-term storage of native specimens of peripheral blood (PB) or bone marrow (BM) and/or the method of RNA isolation has significant influence on gene expression due to gene induction, repression and RNA degradation. The objective of the current study was to investigate the influence of a newly developed RNA stabilization and preparation system for BM specimens (PAXgeneTM Bone Marrow RNA System), focusing on RNA-yield, RNA-integrity and stability of gene expression profiles over time of BM sample storage. Patients and methods: 180 RNA samples that have been processed from 45 heparinized BM specimens of unselected children with acute leukemia at initial diagnosis, during treatment or without pathological findings in hematopoiesis were analyzed. Immediately after collection, heparinized BM specimens were divided into two PAXgeneTM RNA tubes and two standard tubes, each. RNA isolation using either the PAXgeneTM protocol (P) or a reference protocol (R) was performed after 2 hours (P0 and R0) or after 48 hours (P2 and R2). Results: The overall RNA yield (normalized to 1×107 leukocytes) was not different in all four isolation procedures, however the integrity of the RNA using the PAXgeneTM system was significantly higher at both time-points than that of the reference system: 8.6±0.2 (P0) vs. 6.8±0.4 (R0), p=0.0003 and 8.1±0.2 (P2) vs. 6.7±0.5 (R2), p=0.008. For the stabilized samples, we found very good pairwise correlation in gene expression for either gene at P2 compared to P0: GATA1 89±6%, RUNX1 83±10%, NCAM 69±12% and SPI1 89±6%. However, there were significant differences in two of the analyzed genes using the reference RNA isolation procedure: 40±6%, p<0.001 (GATA1) and 47±8%, p=0.005 (NCAM), respectively. Comparing the two RNA isolation procedures, there are significant differences in the expression levels of the analyzed genes. Discussion: The PAXgeneTM system is appropriate for the stabilization of gene expression levels in BM samples after short-term storage. However, as in some genes the reference RNA isolation procedure resulted in similar gene expression levels, the use of a stabilization reagent has to be carefully evaluated within the preanalytical handling of the samples. Our analysis has shown that it is not suitable to compare RNA expression levels derived from different isolation procedures. In conclusion, the PAXgeneTM system is able to stabilize RNA from clinical BM samples while being suitable to isolate high quality and quantity RNA.

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Quantification of MDM2 Gene Expression Level by RQ-PCR Is Predictive of the First Complete Remission in Adulte Acute Myeloid Leukaemia.

Blood ◽

10.1182/blood.v104.11.1068.1068 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1068-1068 ◽

Cited By ~ 1

Author(s):

Nathalie Gachard ◽

Pascal Turlure ◽

Franck Trimoreau ◽

Stephane Moreau ◽

Magali Donnard ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Complete Remission ◽

P53 Gene ◽

Low Doses ◽

Mdm2 Gene ◽

Expression Levels ◽

Mdm2 Expression ◽

Gene Expression Levels ◽

Acute Myeloid

Abstract The overall survival of patients with p 53 mutated gene acute myeloid leukaemia (AML) is significantly shorter than those of patients without mutations. The frequency of p53 gene mutations in AML ranges from 4 to 15% in populations from USA and Europe, thus this is not a useful criteria in the vast majority of patients with a poor prognosis. Mdm2 is the natural functional inhibitor of p53, but very few studies have assessed its prognosis value in AML, and this was done only at the protein level, suffering major lacking of precision. In this study, we have quantified gene expression levels of both MDM2 and p53 by RQ-PCR in a series of 18 patients with more that 50% blast cell in the bone marrow: 4 AML1, 6 AML2 (one with multilineage dysplasia), 4 AML4 (one AML4 Eos), 4 AML5. Median age was 57 years, ranging 39–84. Eight patients had a white cell blood count over 30 G/L. Eleven patients had a normal karyotype, 3 had a complex caryotype, 2 had an inv(16). FLT3 amplification was found in 2 cases. Two patients died immediately following the diagnosis because of an immediate massive global failure of vital functions. Three patients were treated with low doses of Aracytine because of the performance status. Thirteen patients were treated with intensive chemotherapy according classical scheme, and complete remission was obtained in 6 cases. After total RNA extraction of bone marrow mononuclear cells, assessment of MDM2 and p53 gene expression levels was done in duplicate on an ABI PRISM 7000 automat using the TaqManR Assay on demandTM gene expression reference system. The Abl1 gene was used as a reference gene for the control of amplification. The relative expression levels of the genes were calculated as follow: briefly, for each gene, the cycle threshold (CT) was defined for a delta Rn of 10−1. Then, the delta CT (DCT) was calculated as the CT of the gene minus the CT of Abl1 (expected CT for Abl1 ranged from 22 to 24). For each experimental condition, the delta delta CT (DDCT) was calculated as the DCT of the given condition minus the DCT of the RNA pool. For each gene and in each experimental condition, the calculated relative gene expression level was equal to 2−DDCT. No correlation was found between p53 gene expression levels and any of the other clinical, biological or genetic prognosis marker. By contrast, complete remission was obtained for all patients with low levels MDM2 expression and MDM2 expression was markedly increased in 5 out the 7 patient without remission (t-test, p=0,01). It is noteworthy that, among the 3 patients treated with low doses of Aracytine, the patient with the shortest survival was the only one with increased levels of MDM2. No correlation was found between MDM2 gene expression levels and other biological prognosis markers. This suggests that quantification of MDM2 mRNA by RQ-PCR could be an interesting prognosis factor in AML.

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Alpha-5 Integrin Mediates Simvastatin-Induced Osteogenesis of Bone Marrow Mesenchymal Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20030506 ◽

2019 ◽

Vol 20 (3) ◽

pp. 506 ◽

Cited By ~ 7

Author(s):

Pei-Lin Shao ◽

Shun-Cheng Wu ◽

Zih-Yin Lin ◽

Mei-Ling Ho ◽

Chung-Hwan Chen ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Protein Expression ◽

Osteogenic Differentiation ◽

Calcium Deposition ◽

Marker Genes ◽

Expression Levels ◽

Gene Expression Levels

Simvastatin (SVS) promotes the osteogenic differentiation of mesenchymal stem cells (MSCs) and has been studied for MSC-based bone regeneration. However, the mechanism underlying SVS-induced osteogenesis is not well understood. We hypothesize that α5 integrin mediates SVS-induced osteogenic differentiation. Bone marrow MSCs (BMSCs) derived from BALB/C mice, referred to as D1 cells, were used. Alizarin red S (calcium deposition) and alkaline phosphatase (ALP) staining were used to evaluate SVS-induced osteogenesis of D1 cells. The mRNA expression levels of α5 integrin and osteogenic marker genes (bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), collagen type I, ALP and osteocalcin (OC)) were detected using quantitative real-time PCR. Surface-expressed α5 integrin was detected using flow cytometry analysis. Protein expression levels of α5 integrin and phosphorylated focal adhesion kinase (p-FAK), which is downstream of α5 integrin, were detected using Western blotting. siRNA was used to deplete the expression of α5 integrin in D1 cells. The results showed that SVS dose-dependently enhanced the gene expression levels of osteogenic marker genes as well as subsequent ALP activity and calcium deposition in D1 cells. Upregulated p-FAK was accompanied by an increased protein expression level of α5 integrin after SVS treatment. Surface-expressed α5 integrin was also upregulated after SVS treatment. Depletion of α5 integrin expression significantly suppressed SVS-induced osteogenic gene expression levels, ALP activity, and calcium deposition in D1 cells. These results identify a critical role of α5 integrin in SVS-induced osteogenic differentiation of BMSCs, which may suggest a therapeutic strategy to modulate α5 integrin/FAK signaling to promote MSC-based bone regeneration.

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85 Influence of Short-Term Storage on Gene Expression of Equine Embryos

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab85 ◽

2018 ◽

Vol 30 (1) ◽

pp. 182

Author(s):

G. D. A. Gastal ◽

D. Scarlet ◽

R. Ertl ◽

C. Aurich

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Embryo Development ◽

Geometric Mean ◽

Rna Extraction ◽

Short Term ◽

Expression Of Genes ◽

Short Term Storage ◽

Term Storage ◽

Fresh Embryos

Short-term storage for embryo transportation is a routine procedure in equine embryo transfer programs. The success rate after transfer of stored embryos varies among different protocols when embryos are transported overnight. To the best of our knowledge, there is no study evaluating the effect of different temperatures during storage for 24 h on gene expression of equine embryos. Therefore, this study aimed to evaluate the effects of storage of equine embryos for 24 h at 2 temperatures (20°C or 5°C) on the expression of genes related to embryo development (IGF2, H19, POU5F1, SOX2), and regulation of DNA methylation (DNMT1, DNMT3a, DNMT3b). Embryos (n = 24) were collected on Day 7 (n = 18) or Day 8 (n = 6) after ovulation and assigned to 4 groups: Day 7 control (D7, fresh); Day 7, 24 h at 5°C (E5C); Day 7, 24 h at 20°C (E20C); and Day 8 control (D8, fresh 24-h time control). After flushing, embryos were washed and kept in holding medium (Minitube, Tiefenbach, Germany) for morphological classification and measurements. Fresh and stored embryos were treated with pronase (10 mg mL−1), washed with PBS solution and placed in RLT Lysis buffer (Qiagen, Hilden, Germany) for RNA extraction. Total RNA was extracted from each individual embryo using the RNeasy mini kit (Qiagen) following the recommended protocol for animal tissues. After RNA purification, RNA quality was assessed and quantified. Subsequently, cDNA synthesis was performed for RT-qPCR analysis. Two replicates were performed and the relative gene expression was calculated using the 2(–delta delta CT) method, with the target gene expression levels normalized to the geometric mean of PSMB4/SNRPD3. The software SPSS v.24 (IBM/SPSS, Armonk, NY, USA) was used for statistical analyses using the nonparametric tests Kruskal-Wallis and Mann-Whitney U-test to compare differences among groups. Embryos sizes differed (P < 0.05) between D7 (431 ± 48 mm) and D8 (1114 ± 205 mm). Storage temperature did not affect (P > 0.05) embryo size. The mRNA expression of H19 and IGF2 was similar (P > 0.05) among all groups. Expression of POU5F1 and SOX2 was higher (P < 0.05) in D7 and E5C embryos compared with D8 embryos. In addition, E20C had similar (P > 0.05) expression of POU5F1 with D7, E5C, and D8, but lower (P < 0.05) expression of SOX2 when compared with D7. Expression of DNMT1 and DNMT3a were similar (P > 0.05) among D7, D8, and E5C, but lower (P < 0.05) in E20C. Furthermore, expression of DNMT3b was lower (P < 0.05) in D8 and E20C embryos compared with D7 and E5C. In conclusion, temperature during short-term storage seems not to affect the expression of IGF2 and H19 but influences the expression of POU5F1, SOX2, DNMT1, DNMT3a, and DNMT3b. Therefore, these findings suggest that embryos stored at 20°C sustain the pattern of gene expression similar to that of fresh embryos at Day 8, whereas embryos stored at 5°C maintain the gene expression similar to that of fresh embryos at Day 7. Thus, alterations caused by temperature during short-term storage on the expression of genes related to embryo development and DNA methylation may modify the pattern of equine embryonic tissue development, requiring further investigation.

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Hematological Changes in Dogs with Visceral Leishmaniasis Are Associated with Increased IFN-γ and TNF Gene Expression Levels in the Bone Marrow

Microorganisms ◽

10.3390/microorganisms9081618 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1618

Author(s):

Valter Almeida ◽

Isadora Lima ◽

Deborah Fraga ◽

Eugenia Carrillo ◽

Javier Moreno ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Visceral Leishmaniasis ◽

Peripheral Blood ◽

Cytokine Gene Expression ◽

Cytokine Gene ◽

Expression Levels ◽

Ifn Γ ◽

Hematological Changes ◽

Gene Expression Levels

Visceral leishmaniasis is associated with a variety of hematological abnormalities. In this study, we correlated the hematological changes in the peripheral blood of dogs naturally infected with Leishmania infantum (L. infantum) with the distribution of cell lineages and cytokine gene expression patterns in the bone marrow. Samples from 63 naturally semidomiciled dogs living in an endemic area of visceral leishmaniasis were analyzed. L. infantum infection was detected in 50 dogs (79.3%). Among those, 18 (32%) had positive splenic cultures and showed more clinical signs. They also had lower red blood cell counts and leukocytosis with an increased number of neutrophils and monocytes in peripheral blood compared to dogs negative to this test. L. infantum DNA was detected in the bone marrow of 8/14 dogs with positive splenic culture. Dogs with L. infantum infection in the bone marrow presented with histiocytosis (p = 0.0046), fewer erythroid cell clusters (p = 0.0127) and increased gene expression levels of IFN-γ (p = 0.0015) and TNF (p = 0.0091). The data shown herein suggest that inflammatory and cytokine gene expression changes in bone marrow may contribute to the peripheral blood hematological changes observed in visceral leishmaniasis.

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FBN-1, FN-1 and TIMP-3 gene expression levels in patients with thoracic aortic aneurysm

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0305 ◽

2019 ◽

Vol 45 (3) ◽

pp. 263-270

Author(s):

Hülya Özdemir ◽

Sadrettin Pençe ◽

Burcu Çaykara ◽

Hani Alsaadoni ◽

Ender Çoşkunpınar ◽

...

Keyword(s):

Gene Expression ◽

Aortic Aneurysm ◽

Fasting Blood Glucose ◽

Age At Onset ◽

Rna Isolation ◽

Aortic Aneurysms ◽

Tissue Inhibitors Of Metalloproteinases ◽

Tissue Samples ◽

Expression Levels ◽

Gene Expression Levels

AbstractBackgroundAortic aneurysm occurs in the thoracic and abdominal sections of the aorta and is a deadly late-age-at-onset disease. Thoracic aortic aneurysms (TTAs) are characterized by progressive smooth muscle cell rarefaction due to impaired extracellular matrix. The aim of this study was to investigate fibrillin-1 (FBN-1), fibronectin-1 (FN-1) and tissue inhibitors of metalloproteinases-3 (TIMP-3) gene expression levels in patients with TTA.Materials and methodsThe data were analyzed for 16 patients treated for TAA and nine control subjects. Tissue samples obtained during surgery were frozen immediately in liquid nitrogen and stored at −80°C until RNA isolation. Gene expression analysis was performed by quantitative reverse transcription polymerase chain reaction for each gene and Beta actin was used as control gene. 2−ΔΔCT method was used for the determining expression levels of the genes.ResultsAccording to the results of this study, TIMP-3 gene was nine-fold higher expressed in TAA tissues (p = 0.034). Furthermore, TIMP-3 expression levels were found associated with fasting blood glucose, red blood cells and ejection fraction. The gene expression levels of FBN-1 and FN-1 were not statistically significant (p > 0.05).ConclusionIn this clinical trial, we concluded that TIMP-3 expression increases in dilated aorta.

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Selection of reliable reference genes for the normalisation of gene expression levels following time course LPS stimulation of murine bone marrow derived macrophages

BMC Immunology ◽

10.1186/s12865-017-0223-y ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 6

Author(s):

Akane Tanaka ◽

Joyce To ◽

Bronwyn O’Brien ◽

Sheila Donnelly ◽

Maria Lund

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Reference Genes ◽

Time Course ◽

Expression Levels ◽

Murine Bone Marrow ◽

Gene Expression Levels ◽

Selection Of ◽

Stimulation Of

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Differential Modulation of Arcuate Nucleus and Mesolimbic Gene Expression Levels by Central Leptin in Rats on Short-Term High-Fat High-Sugar Diet

PLoS ONE ◽

10.1371/journal.pone.0087729 ◽

2014 ◽

Vol 9 (1) ◽

pp. e87729 ◽

Cited By ~ 15

Author(s):

José K. van den Heuvel ◽

Leslie Eggels ◽

Eric Fliers ◽

Andries Kalsbeek ◽

Roger A. H. Adan ◽

...

Keyword(s):

Gene Expression ◽

Arcuate Nucleus ◽

Differential Modulation ◽

High Fat ◽

Short Term ◽

Expression Levels ◽

High Sugar ◽

Gene Expression Levels

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Prognostic Significance of WT1 and PRAME Gene Expression in Bone Marrow Samples of MDS and AML Patients Treated with Azacytidine

Blood ◽

10.1182/blood.v124.21.5599.5599 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5599-5599

Author(s):

Claire N Andrews ◽

Karen Murphy ◽

Sinead Moran ◽

Patrick Thornton ◽

Nichola Harten ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Overall Survival ◽

Patient Group ◽

Control Group ◽

Prognostic Impact ◽

Wt1 Gene ◽

Transcript Levels ◽

Expression Levels ◽

Gene Expression Levels

Abstract Introduction: The Wilms’ tumor gene (WT1) and preferentially expressed antigen of melanoma (PRAME) gene are frequently overexpressed in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) Combined quantitation of PRAME and WT1 transcript levels could provide a powerful molecular marker in newly diagnosed MDS patients, enabling the early identification of relapse through minimal residual disease monitoring and also to enable alternate treatment strategies (Qin et al, 2012). In this study, we investigated the prognostic impact of WT1 and PRAME gene expression on MDS and AML patients treated with azacytidine. Methods: In this ethically approved, retrospective study, we studied WT1 and PRAME gene expression in the bone marrow of 20 patients with MDS and AML (unsuitable for more intensive chemotherapy) prior to starting epigenetic therapy with azacytidine (100mg/M2 sc for 5 days every 28 days). 20 patients with lymphoma not involving the bone marrow were used as a control group. Quantitative assessment of WT1 and PRAME transcript levels was performed on formalin fixed paraffin-embedded trephine biopsies, using the WT1 Ipsogen ProfileQuant and a ‘homebrew’ kit respectively. A control GADPH was used. Overall survival was extimated using the Kaplan-Meier method and compared using the log-rank test. The logistic regression model was used to correlate IPSS-R with WT-1 and PRAME gene expression levels. Results: The median age of the patient group was 71. 16 patients had MDS (RCMD: 3; RARS: 2; RAEB1: 2; RAEB2: 5; CMML: 4) and 4 had AML. Median age of the control group was 61. Median number of cycles of azacytidine was 4 (range 1-21). In 11 of the patient group and in all of the controls, neither PRAME nor WT1 gene expression was detected.. PRAME gene expression without WT1 gene expression was detected in 5 patients, whilst, in the remaining 4 patients, both PRAME and WT1 gene expression was detected. There was no significant difference in mean percentage of bone marrow blasts between patients with detectable PRAME +/- WT1 gene expression (16.67%) and those without (12.64%). Median gene expression levels of PRAME +/- WT1 significantly correlated with IPSS-R (p=0.003). Overall survival (OS) was significantly inferior in patients who were positive for PRAME +/- WT1 (p=0.0103). Median OS for patients positive for both PRAME and WT1 was 270 days and for patients who were PRAME positive and WT1 negative was 300 days, whilst patients negative for both had a median OS of 630 days (figure 1). Conclusions: Our study results show that patients who have detectable WT1 and PRAME expression in their bone marrow prior to azacytidine therapy have more advanced disease in terms of IPSS-R score and have a much inferior OS compared to patients without detectable WT1 and PRAME. Such patients should be considered for either additional or alternate therapy to single agent azacytidine. Disclosures No relevant conflicts of interest to declare.

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Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

Clinical Chemistry ◽

10.1373/clinchem.2006.078592 ◽

2007 ◽

Vol 53 (4) ◽

pp. 587-593 ◽

Cited By ~ 15

Author(s):

Claudia Langebrake ◽

Kalle Günther ◽

Jürgen Lauber ◽

Dirk Reinhardt

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Gene Expression Pattern ◽

Rna Isolation ◽

Basic Research ◽

Rna Degradation ◽

Rna Integrity ◽

Pairwise Correlation ◽

Mrna Stabilization ◽

Rna Stabilization

Abstract Background: Gene expression profiling is a useful tool for cancer diagnosis and basic research. A major limitation is that, even during short-term storage of native specimens of peripheral blood or bone marrow (BM) and/or RNA isolation, significant changes of gene expression pattern can occur because of gene induction, repression, and RNA degradation. Methods: We investigated the effectiveness of a newly developed RNA stabilization and preparation system for BM specimens (PAXgene™ Bone Marrow RNA System) over time. We analyzed 256 RNA samples, processed from 64 BM specimens. Results: Although the overall RNA yield (normalized to 1 × 107 leukocytes) was not different, the RNA preparation using unstabilized reference samples had an ∼3 times higher failure rate. With the PAXgene system, we observed significantly higher RNA integrity compared with the reference RNA preparation system (P <0.01). In the stabilized samples, we found very high pairwise correlation in gene expression (ΔΔCT 0.16–0.53) for the analyzed genes (GATA1, RUNX1, NCAM1, and SPI1) after 48-h storage compared with immediate preparation of RNA (2 h after BM collection). However, we found major differences in half of the analyzed genes using the reference RNA isolation procedure (ΔΔCT 1.07 and 1.32). Conclusions: The PAXgene system is able to stabilize RNA from clinical BM samples and is suitable to isolate high-quality and -quantity RNA.

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