Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.The research was granted by São Paulo Research Foundation (FAPESP; grant numbers 2009/00938-3, 2010/13384-3, 2010/09561-7, 2011/14207-0 and 2012/07510-1), National Counsel of Technological and Scientific Development (CNPq; grant number 476229/2011-1) and Coordination for the Improvement of Higher Level Personnel (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Induced pluripotent stem cells derived from patients with mitochondrial diseases as a model for studying mitochondrial inheritance

Mitochondrial dysfunctions caused by mutations in the mitochondrial DNA (mtDNA) represent an important group of human pathologies. However, it is not possible to predict with accuracy the risk of a woman with mutant mtDNA to transmit her pathology to her descendants. This is mainly due to out limited understanding of the molecular basis of mitochondrial inheritance. Since development of a technology that enabled derivation of induced pluripotent stem cells (iPSCs) from in vitro culture of somatic cells, iPSCs have become an interesting model to study mitochondrial inheritance. Derivation of iPSCs from patients with pathogenic mtDNA mutations has revealed that the mutant load decreases through in vitro culture of iPSCs, suggesting the existence of a specific mechanism that eliminates mutant mtDNA in the germ line. Thus, the aim of this work was to use iPSCs derived from patients with mitochondrial disorders to investigate the existence of a mechanism that eliminates mtDNA molecules with pathogenic mutations. In this way, we used heteroplasmic fibroblasts harboring a point mutation A3243G in mtDNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS); heteroplasmic fibroblasts harboring a deletion in mtDNA causing Kearn-Sayre Syndrome (KSS) and homoplasmic fibroblasts containing only wild-type mtDNA (Control). The KSS lineage derivation resulted in iPSCs with low levels of mutant mtDNA (<0,1%), and the elimination of mutant molecules during the culture. The MELAS derivation resulted in iPSCs with high levels of mutant mtDNA (> 98%), and indication of mutant molecules elimination as well. However, unexpectedly, there was no reduction of mtDNA content in iPSCs compared to fibroblasts in all lineages. On contrary, mtDNA copy number increased in MELAS and KSS iPSCs, perhaps due to the high levels of mutations in the cells. No effect of Rapamycin (mitophagy inductor) treatment was detected on the yield of colony formation in MELAS iPSCs. Additionally, Rapamycin did not affect the mutation levels in MELAS iPSCS compared to untreated iPSCs. Finally, gene expression analysis of MELAS iPSCs provided evidences of an autophagic mechanism directed towards the mitochondrion.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Disfunções mitocondriais causadas por mutações no DNA mitocondrial (mtDNA) representam um importante grupo de patologias humanas. No entanto, não é possível predizer com acurácia o risco de uma mulher acometida por uma mutação no mtDNA transmitir a patologia para seus descendentes. Isso se deve, em parte, ao desconhecimento dos mecanismos moleculares que controlam a herança mitocondrial. Com o desenvolvimento de metodologias que possibilitam a derivação de células pluripotentes induzidas (iPSCs) a partir de células somáticas cultivadas in vitro, as iPSCs se tornaram um interessante modelo para o estudo da herança mitocondrial. A derivação de iPSCs de pacientes com mutações patogênicas no mtDNA tem revelado que a porcentagem de moléculas mutantes diminui ao longo do cultivo, sugerindo a existência na linhagem germinativa de mecanismos específicos para eliminação de mtDNAs mutantes. Portanto, o presente trabalho investigou em iPSCs derivadas de pacientes com desordens mitocondriais a existência de um mecanismo celular que elimina as moléculas de mtDNA com mutações patogênicas. Para tanto, foram utilizados fibroblastos heteroplásmicos portadores da mutação pontual A3243G no mtDNA causadora de encefalomiopatia mitocondrial, acidose lática e episódios tipo acidente vascular cerebral (MELAS); fibroblastos heteroplásmicos portadores de uma deleção de 4,9 kb no mtDNA causadora da Síndrome de Kearns-Sayre (KSS) e fibroblastos Controle, contendo apenas mtDNA selvagem. A derivação de linhagens portadoras de KSS resultou em iPSCs com baixos níveis de mtDNA mutante (< 0,1%), e na eliminação de moléculas mutantes ao longo do cultivo. A derivação de linhagens portadoras de MELAS resultou em iPSCs com alta taxa de mutação (> 98%), também com indícios de diminuição da quantidade de moléculas mutantes ao longo do cultivo. No entanto, ao contrário do esperado, não houve diminuição da quantidade de cópias de mtDNA nas iPSCs em relação aos fibroblastos em todas as linhagens (Controle, KSS e MELAS), sendo que as iPSCs de MELAS e KSS apresentaram um aumento significativo na quantidade de cópias de mtDNA, provavelmente devido a efeitos causados pela mutação no mtDNA. Ao analisar o efeito do tratamento com Rapamicina (indutor de mitofagia) durante a derivação de MELAS não observamos aumento na eficiência de formação de colônias, além de o tratamento não afetar a quantidade de mtDNA mutante, resultando em iPSCs com níveis de mutação similares aos encontrados nas iPSC MELAS não tratadas com o rapamicina. Por fim, resultados de expressão gênica das iPSCs do grupo MELAS revelaram indícios de mecanismos autofágicos direcionados a mitocôndria provavelmente devido ao efeitos causados pela a alta taxa da mutação.2013/13869-

Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

<div><p>Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.</p></div

Level of NZB mtDNA across generations in a heteroplasmic mouse lineage produced by cytoplasmic transfer.

<p>Circles depict the level of NZB mtDNA in mice estimated by ARMS-qPCR from ear biopsies. Filled circles depict females that were backcrossed (BC) with B6 males to obtain the next generation progeny (BC₁, BC₂, BC₃, BC₄ and BC₅). The level of NZB mtDNA was not determined for males, except in the founder lineage where males are depicted by empty circles. In the remaining generations (BC₁, BC₂, BC₃, BC₄ and BC₅) empty circles depict females that were not mated. Bars represent the means.</p

Specificity of the ARMS-qPCR approach for amplification of target mtDNA.

<p>Using samples from homoplasmic mice (either NZB or B6), non-specific amplification of non-target mtDNA accounted for only 0.01% of target amplification. Analysis of samples from heteroplasmic mice showed that levels as low as 0.1% of mtDNA of NZB or B6 origin could be detected. Individual Ct values (cycle at which plots crossed the threshold) are denoted in the inset.</p

Comparison of mtDNA copy number among tissues of heteroplasmic males.

<p>Bars depict mtDNA copy number per cell from mice aged four months. Tissues analyzed included brain, heart, liver and tail. Values are reported as mean ± SEM. Bars with different letters denote a significant difference among tissues (P < 0.05).</p

Analysis of primer efficiency.

<p>Standard curves were generated by qPCR using as template 5-fold serial dilutions of heteroplasmic gDNA or pDNA (25, 5, 1 and 0.2 ng per reaction). First-degree linear regressions were fitted for the log of input amount of template versus the Ct (A) or the ∆Ct (B) values for serial-diluted DNAs. The ∆Ct was calculated by subtracting either Ct_B6 from Ct_NZB (heteroplasmy quantification) or Ct_mtDNA from Ct_Apob (copy number quantification). Comparison of amplification efficiency between primer pairs (ARMS2/MT14 vs. ARMS22/MT20 and MT14/MT15 vs. OIMR1544/OIMR3580) was found similar as the slope values from linear regressions were equal or smaller than 0.1 (B). Moreover, the use of gDNA or pDNA for heteroplasmy quantification did not affect slope values.</p

Primer sequences used for measuring heteroplasmy and mtDNA copy number.

<p>^aUnderlined nucleotides are complementary to one of mtDNA haplotypes (NZB or B6) due to the presence of a polymorphic nucleotide at position 3,599 (ARMS22) and 3,932 (ARMS2), respectively. A mismatched nucleotide introduced immediately 5’ to the polymorphic sites is represented in lowercase.</p><p>^bNon-driscriminative assays that amplify mtDNA of B6 and NZB origin.</p><p>^cDriscriminative assay that amplifies mtDNA of B6 origin.</p><p>^dDriscriminative assay that amplifies mtDNA of NZB origin.</p><p>Primer sequences used for measuring heteroplasmy and mtDNA copy number.</p

Relationship between the input level of NZB gDNA and the level of NZB mtDNA estimated by ARMS-qPCR.

<p>Different proportions of gDNA from NZB mice (100%, 99.9%, 99.5%, 99%, 95%, 90%, 75%, 50%, 25%, 10%, 5%, 1%, 0.5%, 0.1% and 0%) were mixed with gDNA from B6 mice to be analyzed by ARMS-qPCR. Filled circles depict the use of 0.5 ng of DNA per reaction (duplicates) whereas empty circles depict the use of 5.0 ng of DNA (triplicates). The relationship between the input level of NZB gDNA and the level of NZB mtDNA determined by ARMS-qPCR was analyzed by calculating Pearson’s correlation coefficient (r). P value is denoted in the inset.</p