Computational protocol: Persistent fibrosis, hypertrophy and sarcomere disorganisation after endoscopy-guided heart resection in adult Xenopus

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Protocol publication

[…] For RT-qPCR, independent biological replicates were collected from CTRL, SHAM and AMP animals. Hearts were collected, cleaned from atrium and arterial bulb (only the ventricle part was kept), then cut in half and thoroughly washed in PBS to eliminate the blood (which might compete with heart tissue gene expression as blood cells are nucleated), before being snap frozen in liquid nitrogen. Total RNAs were extracted from snap-frozen adult Xenopus heart ventricles using RNAqueous® Phenol-free total RNA isolation kit (Ambion), then quantified and quality controlled using Qubit (Thermo Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies) respectively. Samples with RNA integrity (RIN) of ≥7 (average RIN 8.6) were reverse transcribed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) using a MyCyclerTM (Bio-Rad), following manufacturers recommendations. RT-qPCR reactions were performed in duplicate for each sample using Power SYBR® master mix on a QuantStudioTM6 Flex Real-Time PCR System (Applied Biosystems), following manufacturer recommendations. Primers (MWG Biotech) were designed using Primer Blast [] and the relevant sequences are listed in . Ct data were collected using ExpressionSuite Software (Applied Biosystems) and analysed using Excel (Microsoft). The Cts for each technical duplicate were averaged and normalised (ΔCt) against the geometric mean of two reference genes (smarcd1 and smn2), which were chosen after analysis by the NormFinder software []. These normalisers were chosen among 12 putative normalisers, already known to be expressed in adult Xenopus laevis cardiac ventricles (actnl6a, actn3, akt1, cebpb, col1a1, cxcl8, fn1, hif1a, smarcd1, tert; our unpublished data) or to be good normalisers in other tissues (smn2, odc); they were ranked on the basis of their expression stability in the samples presented in this paper and the best pair was retained. Variations of expression were quantified by the ΔΔCts method [], using the SHAM condition as references for each experimental time-points and fold changes were computed as 2-ΔΔCt. Statistical analyses were performed on ΔCts with Prism 7 (GraphPad). Outliers were removed with ROUT test method (Q = 1%). One-Way ANOVA followed by Holm-Sidak’s post-test were performed for datasets that passed a Shapiro-Wilk normality test; Kruskal-Wallis non-parametric test followed with Dunn's post-tests were performed for those that did not pass this test (namely fn1, 3 dpa; col1a1, 7 dpa; nppb, 7dpa; actn3, 3 dpa; tnnt2, 1 dpa; cxcl8, 7 dpa). *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001). […]

Pipeline specifications

Software tools Primer-BLAST, NormFinder
Application qPCR
Organisms Homo sapiens, Xenopus laevis, Danio rerio, Mus musculus, Oryzias latipes
Diseases Heart Diseases