Computational protocol: A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct

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Protocol publication

[…] Total cellular RNA isolation from three independent CCC cultures of various combinations (vasculogenic: hCMVECs culture, hCMVECs/hMSCs co-culture; cardiomyogenic: hiPSC-ECMs culture, hiPSC-ECMs/hMSCs co-culture) that were maintained either in vasculogenic medium or myogenic medium were done using TRIzol® Plus RNA purification kit (Invitrogen) as per manufacturer's instructions.The RNA integrity (RIN) of the extracted samples was analyzed on the Agilent 2100 Bioanalyzer system using the Agilent RNA 6000 nano kit (Agilent Technologies, Inc.) following the manufacturer's recommendations. The reverse transcriptase (RT) reaction was executed using 250 ng of total RNA in a final reaction volume of 20 μl using an iScript™ Reverse Transcription Supermix for RT-qPCR kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocols.The cardiomyogenic gene-specific primers for MYH6 (myosin heavy chain 6), MYH7 (myosin heavy chain 7), ACTC1 (actin, alpha, cardiac muscle 1), TNNI3 (troponin I3, cardiac type), GATA4 (GATA binding protein 4), NPPA (natriuretic peptide A), NPPB (natriuretic peptide B), and GJA1 (gap junction protein, alpha 1); and the vasculogenic gene-associated primers for PECAM1 (platelet and endothelial cell adhesion molecule 1), KDR (kinase insert domain receptor, a type III receptor tyrosine kinase), TIE1 (tyrosine kinase with immunoglobulin-like and EGF-like domains 1), TEK (TEK tyrosine kinase, endothelial), and VWF (von Willebrand factor); as well as the endogenous normalizer reference genes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-ACTIN (cytoplasmic beta-actin), G6PD (glucose-6-phosphate dehydrogenase, and RPLP0 (ribosomal protein lateral stalk subunit P0) were designed using web based software Primer3 (Rosen and Skaletsky, ), synthesized commercially (Integrated DNA Technologies, Inc.), and evaluated for an uniform annealing temperature of 58°C, for all the primer pairs, as shown in Table .Real-time PCR conditions were optimized as described previously (Valarmathi et al., ,; Willems et al., ; Bustin et al., ). All RT-qPCRs were performed with SsoAdvanced™ SYBR® Green Supermix in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.), and CT (threshold cycle) values were calculated using the CFX Manager™ software, Security Edition. The calibrator control included hCMVECs day 0 sample for vasculogenic cultures and hiPSC-ECMs day 0 sample for cardiomyogenic cultures, and the target gene expression was normalized by three non-regulated reference gene expressions, viz., GAPDH, β-ACTIN, and either G6PD or RPLP0. The expression ratio of genes was determined by applying the mathematical model previously described by Pfaffl et al. (). [...] The RT-qPCR experimental data were represented as mean ± standard error of the mean (mean ± SEM). The differences in expression profile (cardiomyogenic and vasculogenic markers) between control (day 0) and treated samples (day 7 or 14) were determined in-group means for statistical significance by applying ‘Pair Wise Fixed Reallocation Randomization Test’ using Relative Expression Software Tool—384 (REST-384- version 2) (Pfaffl et al., ). In all cases, p < 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools Primer3, CFX Manager, REST
Application qPCR
Organisms Homo sapiens
Diseases Vascular Diseases
Chemicals Oxygen