Identification of valid housekeeping genes for real-time quantitative PCR analysis of collapsed lung tissues of neonatal somatic cell nuclear transfer-derived cattle Liu, Yan; Zhang, Yunhai; Jiang, Qiuling; Rao, Man; Sheng, Zheya; Zhang, Yu; Du, Weihua; Hao, Haisheng; Zhao, Xueming; Xu, Zhe; Liu, Jianning; Zhu, Huabin Published in: Cellular Reprogramming DOI: 10.1089/cell.2015.0024 Publication date: 2015 Document version Publisher's PDF, also known as Version of record Citation for published version (APA): Liu, Y., Zhang, Y., Jiang, Q., Rao, M., Sheng, Z., Zhang, Y., Du, W., Hao, H., Zhao, X., Xu, Z., Liu, J., & Zhu, H. (2015). Identification of valid housekeeping genes for real-time quantitative PCR analysis of collapsed lung tissues of neonatal somatic cell nuclear transfer-derived cattle. Cellular Reprogramming, 17(5), 360-367. https://doi.org/10.1089/cell.2015.0024 Download date: 26. sep.. 2021 CELLULAR REPROGRAMMING Volume 17, Number 5, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/cell.2015.0024 Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer–Derived Cattle Yan Liu,1,4 Yunhai Zhang,2,4 Qiuling Jiang,1 Man Rao,3 Zheya Sheng,3 Yu Zhang,2 Weihua Du,1 Haisheng Hao,1 Xueming Zhao,1 Zhe Xu,1 Jianning Liu,1 and Huabin Zhu1 Abstract Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is com- monly adopted in gene expression analysis. However, selection of an appropriate reference gene for normaliza- tion is critical for obtaining reliable and accurate results. Seven housekeeping genes—b-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), b-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)—were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases. Introduction alveolar wall shows deteleotasis under microscopy, and consolidation of lung tissue can be found in dissection. omatic cell nuclear transfer (SCNT) is a powerful In humans, a similar condition called neonatal respiratory Smethod for producing transgenic animals from the same distress syndrome occurs in premature infants and is caused genetic background. The technique involves in vitro manip- by developmental insufficiency of surfactant production ulation and embryonic culture that may lead to inadequate and structural immaturity in the lungs (Avery and Mead, nuclei reprogramming and therefore result in abnormal gene 1959). expression in embryos and animals (Everts et al., 2008; There have been several approaches for investigating Jafarpour et al., 2011; Lin et al., 2008). Developmental de- mechanisms of alveoli collapse in neonatal cloned cattle, ficiency leads to a low survival rate of newborns, especially in most of which focus on imprinted genes and the methylation cloned animals. Respiratory failure resulting from alveoli status of certain transcription factors (Lin et al., 2008). collapse is a high-frequency event and one major cause of However, the underlying pathophysiological mechanisms death in newborn cloned cattle (Hill et al., 1999; Lanza et al., that account for this condition are yet to be elucidated. 2001). Alveoli collapse in neonatal cloned cattle is a condi- Numerous genes are involved in lung development, thus tion accompanied by symptoms such as ventilation defi- identification of the genes responsible for lung collapse in ciency and disturbance of air exchange in the lung tissue, newborn cattle and evaluation of gene expression profiles of leading to dyspnea and even respiratory failure. The pulmonary target genes in collapsed lung tissues are prerequisites. 1Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. 2Anhui Provincial Laboratory of Local Livestock and Poultry Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China. 3Geno-Ming Bioscience, Beijing, 101101, China. 4These authors contributed equally to this work. 360 REFERENCE GENE SELECTION IN COLLAPSED LUNG 361 Real-time quantitative PCR (qPCR) is a powerful tool for RNA extraction, DNase treatment, monitoring changes in gene expression and for understanding and cDNA synthesis biological and molecular mechanisms due to its sensitivity, Total RNA was extracted from lung tissues using the accuracy, specificity, and broad quantification range. To ac- RNeasy Mini Kit (Qiagen, Hilden, Germany) according to quire accurate and meaningful gene expression quantification, the manufacturer’s protocol with DNase treatment during the magnitude of expression normalized to a reference gene as purification of RNA. RNA concentration and purity were an internal control is required. The reference gene is often determined by measuring the absorbance at A260 and A280 referred to as a housekeeping gene (HKG), which should be using a NanoDrop ND-1000 Spectrophotometer (NanoDrop expressed constitutively at relative constant levels under either Technologies Inc., USA). RNA integrity was checked by different experimental conditions or different disease pro- electrophoresis of 500 ng of RNA on 0.8% agarose ethidium cesses. However, commonly used HKGs cannot be universal bromide–stained gels. A mean ratio of A260/A280 &2.0 is under all experimental conditions; indeed, previous studies generally accepted as pure RNA. have shown that some HKGs can vary considerably either in cDNA was synthesized using the QuantiTect Reverse different cell types or under different disease processes (Bustin, Transcription Kit (Qiagen, Hilden, Germany). Briefly, 2 lg 2000; Suzuki et al., 2000; Thellin et al., 1999; Vandesompele of total RNA was used to produce first-strand cDNA with an et al., 2002; Warrington et al., 2000). Therefore, it is crucial to oligo(dT) primer and random primers. seek stably expressed HKGs through a systematic study to interpret the qPCR data more effectively. Primer design and real-time quantitative PCR Few articles in the literature could be found concerning alveolar collapse in cloned cattle; therefore, comparison Oligonucleotide primer pairs were generated by Primer data of suitable HKGs expressed stably in collapsed lung tissue Express 3.0 (Applied Biosystems, Foster City, CA, USA) for quantification is limited. The aim of this study was to according to the published bovine sequences in GenBank identify candidate reference genes for qPCR in the collapsed and synthesized by the Beijing Invitrogen Company (Beijing, lung tissue of cloned cattle. The expression levels of seven China). The primer efficiency was tested through a serial commonly used HKGs—b-glucuronidase (GUSB), phospho- dilution curves with the equation: PCR efficiency = (10[-1/slope] glycerate kinase 1 (PGK1), b-2-microglobolin (B2M), pepti- - 1) $ 100. dylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate qPCR amplification was performed using the 7500 Fast dehydrogenase (GAPDH), TATA-box binding protein (TBP), Real-time PCR system (Applied Biosystems, Foster City, and 5.8S ribosomal RNA (5.8S rRNA)—were assessed in the CA, USA) following the manufacturer’s instructions and collapsedlungtissuesofcloned cattle and normal lungs of age- with SYBR Green Real-Time PCR with ROX correction. matched normal cattle, and the comparison data were analyzed Briefly, 1 lL of 1:10 diluted cDNA, 1 lL of primer pairs through three different statistical methods. (10 pmol/lL for each primer), 7.5 lLof2· Power SYBR Green Real-Time PCR Master Mix (Applied Biosystems, Materials and Methods Foster City), and 5.5 lL of double-distilled water was added to a final reaction volume to 15 lL. The holding conditions for Animals qPCR were as follows: Elimination of carryover contamina- Collapsed lung tissues from four neonatal cloned cattle that tion by uracil N-glycosylase (UNG) at 50°C for 2 min and died of respiratory failure and normal lung tissues from four activation of Taq polymerase and template denaturation at age-matched normal cattle were collected for this study. All 95°C for 10 min. The cycling conditions included template procedures were performed at the Institute of Animal Sciences, denaturation at 95°C for 15 sec and annealing and elongation Chinese Academy of Agricultural Sciences (Beijing, China) at 60°C and 72°C for 1 min, respectively. To ensure a single and were in accordance with the Guiding Principles for the product for each reaction, melt curve analysis was added after Care and Use of Laboratory Animals. thermocycling ranging from 60°Cto95°C by temperature increases of 0.5°C per cycle. All