custom self designed sybr green primers  (Thermo Fisher)

Journal: International Journal of Stem Cells

Article Title: Moderate Hypoxia Exhibits Increased Endothelial Progenitor Vessel-forming Ability However Gestational Diabetes Caused to Impede Compensatory Defense Reaction

doi: 10.15283/ijsc.2016.9.1.152

Figure Lengend Snippet: Real-time PCR reactions were performed for endothelial specific markers ((A) VE-cadherin [CDH5, vascular endothelial-cadherin, CdH5, Ca ++ -dependent cell adhesion molecule, CD144], (B) endothelial nitric oxide synthase [eNOS] and (C) endothelial cell adhesion molecule [PECAM; CD 31]) using “custom designed” SYBR green mix as previously described. The data were analyzed in duplicated using the “modified” 2 −ΔΔCT equation. X axis is representing group of samples; Control Normoxic (C-HUCB ECFC-N, human umbilical cord blood ECFCs obtained from control pregnancies and plated under normoxic condition), Control Hypoxic (C-HUCB ECFC-H, human umbilical cord blood ECFCs obtained from control pregnancies and plated under hypoxic condition), GDM normoxic (GDM-HUCB ECFC-N, human umbilical cord blood ECFCs obtained from GDM subjects and plated under normoxic condition), GDM Hypoxic (GDM-HUCB ECFC-H, human umbilical cord blood ECFCs obtained from GDM subjects and plated under hypoxic condition). Y axis is representing the results of modified” 2 −ΔΔCT . (B) *eNOS mRNA expression in GDM-HUCB ECFC-H significantly decreased if compare with C-HUCB ECFC-N, C-HUCB ECFC-H and GDM-HUCB ECFC-N (p<0.05).

Article Snippet: Real-time PCR reactions were performed for PECAM 1 (CD31), VE-cadherin (CD144), eNOS, VEGFA, IGF-1 and β -actin in duplicated with a custom designed SYBR Green mix [2.4 μ l of 25 mM MgCl 2 , 5 μ l of 1:10,000 dilution SYBR Green I (Molecular Probes) and 5 μ l of 1 nM Fluorescein Calibration Dye (1 mM/L in DMSO, Bio-Rad) in 50 μ l of total reaction using Taq DNA polymerase (Promega) as described previously ( , – )].

Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Modification, Control, Expressing

Journal: International Journal of Stem Cells

Article Title: Moderate Hypoxia Exhibits Increased Endothelial Progenitor Vessel-forming Ability However Gestational Diabetes Caused to Impede Compensatory Defense Reaction

doi: 10.15283/ijsc.2016.9.1.152

Figure Lengend Snippet: Real-time PCR reactions were performed for pro-angiogenic markers ((A) vascular endothelial growth factor A (VEGFA) and (B) insulin-like growth factor 1 [IGF-1]) and hypoxia specific markers ((C) adrenomedullin [ADM], (D) G protein coupled activity modifying protein 2 [RAMP 2]) using “custom designed” SYBR green mix as previously described. The data were analyzed in duplicated using the “modified” 2 −ΔΔCT equation. X axis is representing group of samples: Control Normoxic (C-HUCB ECFC-N, human umbilical cord blood ECFCs obtained from control pregnancies and plated under normoxic condition), Control Hypoxic (C-HUCB ECFC-H, human umbilical cord blood ECFCs obtained from control pregnancies and plated under hypoxic condition), GDM normoxic (GDM-HUCB ECFC-N, human umbilical cord blood ECFCs obtained from GDM subjects and plated under normoxic condition), GDM Hypoxic (GDM-HUCB ECFC-H, human umbilical cord blood ECFCs obtained from GDM subjects and plated under hypoxic condition). Y axis is representing modified” 2 −ΔΔCT . (A) *VEGFA mRNA expression in control HUCB ECFCs which plated in vitro moderate hypoxic environment was found significantly increased if compare with their normoxic controls (p<0.05). (D) *RAMP 2 mRNA expression was found significantly decreased if compare with their normoxic and hypoxic controls (p<0.05).

Article Snippet: Real-time PCR reactions were performed for PECAM 1 (CD31), VE-cadherin (CD144), eNOS, VEGFA, IGF-1 and β -actin in duplicated with a custom designed SYBR Green mix [2.4 μ l of 25 mM MgCl 2 , 5 μ l of 1:10,000 dilution SYBR Green I (Molecular Probes) and 5 μ l of 1 nM Fluorescein Calibration Dye (1 mM/L in DMSO, Bio-Rad) in 50 μ l of total reaction using Taq DNA polymerase (Promega) as described previously ( , – )].

Techniques: Real-time Polymerase Chain Reaction, Activity Assay, SYBR Green Assay, Modification, Control, Expressing, In Vitro