Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos

Lee, Won–Jae; Jang, Sang Ock; Lee, Seung-Chan; Park, Jisung; Jeon, Ryoung-Hoon; Subbarao, Raghavendra Baregundi; Bharti, Dinesh; Shin, Junga; Park, Bong-Wook; Rho, Gyu‐Jin

doi:10.1071/rd14393

Cited by 18 publications

(34 citation statements)

References 53 publications

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“…Although the number of DEGs and fold change values were similar for both developmental stages, vitri ed morulae and blastocysts displayed very different transcriptome pro les, with only 12 (three upregulated and eight downregulated) DEGs in common. This is not surprising, considering that stage-speci c gene expression between morulae and blastocysts is characteristic of the preimplantation embryo development [43].…”

Section: Discussionmentioning

confidence: 95%

Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

Cuello¹,

Martínez²,

Cambra³

et al. 2020

Preprint

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BackgroundDespite reported promising farrowing rates after non-surgical and surgical transfers of vitrified in vivo-derived porcine morulae and blastocysts (range: 70-75%), their pregnancy loss is yet higher (10-20%) than it is for fresh embryos (< 2.5%). The present study was designed to investigate whether vitrification would affect the transcriptome of porcine morulae and blastocysts, using microarrays and qRT-PCR validation. Material and methods and resultsMorulae and blastocysts were collected from weaned sows (n=13) by laparotomy at Day 6 (Day 0=start of estrus). Of each embryo category, 50 morulae and 50 blastocysts were vitrified (Treatment group) while fresh morulae (n=40) and blastocysts (n=40) acted as control group. After one week of storage, vitrified embryos were cultured in vitrofor 24 h after warming. Non-vitrified morulae (n=40) and blastocysts (n=40), cultured in vitro for 24 h were used as controls. After the in vitro culture period, embryo viability was morphologically assessed. A total of 30 viable embryos per group and embryonic stage(three pools of 10), were subjected to gene expressionanalysis byusing a microarray approach. A fold change cut-off of ±1.5 and a p-unadjusted <0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed morulae and blastocysts were similar to those of the control (nearly 100%, n.s.).A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated) and 105 (112 upregulated and 93 downregulated) in vitrified blastocysts compared to the control group.Transcriptome DEG profiles were dependent on developmental stage, and GO enrichment analysis mainly related DEGs tobiological processes.The vitrification/warming impact on morulae was mostly repression of gene expression with the exception of metabolism-related pathways. In the case of blastocysts, we noted the activation of the cell cycle, cellular senescence andof signaling pathways for TFGβ, p53, FoxO and MAPK. Disruption ofmetabolic-related pathways in morulae and steroid biosynthesis and gap junctions in blastocysts could be related to impaired embryo quality and developmental potential, despite the rather high post-warming survival rates seen in vitro.ConclusionIn conclusion, vitrification modified the transcriptome of in vivo-derived porcine morulae and blastocysts, resulting in moderate gene expression changes, which may disturb subsequent embryo development and pregnancy after embryo transfer.

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Section: Discussionmentioning

confidence: 95%

Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

Cuello¹,

Martínez²,

Cambra³

et al. 2020

Preprint

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“…Our results showed that amongst the genes expressing constant patterns (stability) in the human muscle regardless of sex and physiological conditions, the most stable one is the gene of hypoxanthine phosphoribosyl transferase ( HPRT1 ). The establishment of a tissue-specific reference gene can help discriminate both distinctive characteristics, and genetic-based changes in cell functioning among different physiological conditions 36 , 37 , 38 , 39 ; 40 . Precision in the determination of a gene of reference for comparative analysis of the expression of specific proteins is crucial to enable the assessment of factors that are expressed in low quantities in tissue.…”

Section: Discussionmentioning

confidence: 99%

BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR - a Novel Protocol

Assis¹,

Hoffman²,

Gasanov³

2020

Int. J. Med. Sci.

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Background: Alteration in brain-derived neurotrophic factor (BDNF) production is a marker of neuropathological conditions, which has led to the investigation of Val66Met polymorphism occurring in the human BDNF gene ( BDNF ). Presently, there are no reported methods available for the analysis of Val66Met impact on human BDNF functioning. Purpose: To develop a qRT-PCR protocol for the allele-specific expression evaluation of the Val66Met polymorphism in BDNF . Methods: Using RNA extracted from muscle samples of 9 healthy volunteers (32.9 ± 10.3 y) at rest and following a maximal effort aerobic capacity exercise test, a protocol was developed for the detection of Val66/Met66 allele-specific BDNF expression in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) - relative to housekeeping genes - and validated by absolute quantification in Droplet Digital Polymerase Chain Reaction (ddPCR). Results: Differences in the relative values of BDNF mRNA were confirmed by ddPCR analysis. HPRT1 and B2M were the most stable genes expressed in muscle tissue among different metabolic conditions, while GAPDH revealed to be metabolic responsive. Conclusion : Our qRT-PCR protocol successfully determines the allele-specific detection and changes in BDNF expression regarding the Val66Met polymorphism.

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“…Therefore, the selection of nonvalidated or unstable RGs during normalization of GOI may lead to inconsistent or misleading results [11, 37]. The incorrect selection of RGs drastically affects the results [4, 11, 22, 38]. When the expression of lineage-specific markers in differentiated MSCs toward adipocytes, osteoblasts, and chondrocytes was normalized against the most stable RGs (RPLP0 and EEF1A1 for osteogenesis; TBP and YWHAZ for adipogenesis and chondrogenesis) and the least stable RG (EEF1A1 for adipogenesis; B2M for osteogenesis and chondrogenesis), inconsistent results of the relative expression of lineage-specific markers were significantly ( p < 0.0001) identified.…”

Section: Discussionmentioning

confidence: 99%

“…The primers for each RG were designed by Primer 3 Plus software (considering 60°C annealing temperature) and were analyzed for the formation of hairpins, homodimers, and heterodimers using OligoAnalyzer 3.1 software. In order to check the PCR efficiencies, a standard curve of each primer was made from the cycle threshold (Ct) values by a four-fold serial dilution of cDNA (1:10, 1:100, 1:1,000 and 1:10,000) of E-BM-MSCs following the previous protocols [6, 22]. Standard curve parameters such as slope (M), intercept (B), PCR efficiencies (E = 10 -1/slope -1 × 100), and correlation (R 2 ) were calculated using RotorGene Q Series Software (Qiagen, Hilden, Germany) or Excel (Microsoft, WA, USA).…”

Section: Methodsmentioning

confidence: 99%

PPIA, HPRT1, and YWHAZ Genes Are Suitable for Normalization of mRNA Expression in Long-Term Expanded Human Mesenchymal Stem Cells

Jeon

Lee

Son

et al. 2019

BioMed Research International

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Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.

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Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos

Cited by 18 publications

References 53 publications

Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR - a Novel Protocol

PPIA, HPRT1, and YWHAZ Genes Are Suitable for Normalization of mRNA Expression in Long-Term Expanded Human Mesenchymal Stem Cells

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