194 HOUSEKEEPING GENE EXPRESSION OF BOVINE FERTILIZED AND CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 195 ◽  
Author(s):  
P. J. Ross ◽  
K. Wang ◽  
Z. Beyhan ◽  
A. Kocabas ◽  
J. B. Cibelli
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
18S Rrna ◽  
Cyclophilin A ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Blastocyst Stage ◽  
Histone H2a ◽  
Cell Stage ◽  
Expression Levels

Real-time RT-PCR can accurately quantify mRNA levels in pre-implantation embryos; however, comparisons among different embryonic stages and among embryos produced by different means often rely on a control gene, which is commonly assumed to remain constant across samples. The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine pre-implantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). Embryos were produced according to standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). Total RNA was collected from 3 pools of 10 oocyte/embryos at metaphase II (MII), PN, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and reverse-transcribed using oligo-dT primers. The cDNA was then amplified using PCR (Kocabas et al. 2006 PNAS 103, 14 027–14 032). All amplified cDNA samples were diluted to 1 ng μL–1, as determined by NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and corroborated using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). For real-time PCR, 2 μL of cDNA was analyzed in duplicates. Absolute quantification was performed as previously described (Iager et al. 2008 Cloning Stem Cells doi:10.1089), using SYBR-green chemistry and standard curves specific for each gene. The number of RNA copies per nanogram of amplified cDNA was compared among samples using ANOVA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin A, β-actin, ribosomal protein L-15 (RPL-15), and β-glucuronidase (GUS) expression levels were similar in MII oocyte, 1-, 2-, 4-, and 8-cell embryos, while a significant increase at morula and blastocyst stages was observed (P < 0.05). A similar pattern of expression was observed for 18S ribosomal RNA, but with a significant decrease from morula to blastocyst stages (P < 0.05). Histone H2A expression was significantly higher at 1-cell stage, similar from 2-cell to morula stages and lowest at the blastocyst stage. We then compared expression between IVF and SCNT embryos at 2-, 4-, and 8-cell and blastocyst stages. GAPDH, RPL-15, GUS, and β-actin were significantly different among groups in at least 3 of the analyzed stages, which in all cases included blastocysts. 18s-rRNA was different among IVF and SCNT embryos only at the 8-cell stage, while no differences were observed at any stages for histone H2A and cyclophilin A. At the blastocyst stage, the lowest overall variability among IVF and SCNT embryos was observed for 18s-rRNA. In conclusion, none of the evaluated housekeeping genes showed consistent mRNA expression levels across developmental stages of IVF embryos. In addition, SCNT embryos, compared to IVF, had different levels of gene expression for commonly used housekeeping genes, which, if neglected, might result in data misinterpretation. In our conditions, histone H2A had similar expression levels between IVF and SCNT embryos across different stages and showed less variability than cyclophilin A. Finally, for comparisons at the blastocyst stage, 18s-rRNA had the least variability among IVF and SCNT embryos.

51 AGGREGATION REDUCES THE VARIATION OF GENE EXPRESSION LEVELS IN BOVINE CLONES

2006 ◽  
Vol 18 (2) ◽  
pp. 134
Author(s):  
S. Kurosaka ◽  
N. A. Leu ◽  
K. J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Coefficient Of Variation ◽  
Blastocyst Stage ◽  
Average Level ◽  
Cdna Libraries ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Expression Levels ◽  
Gene Expression Levels

Mammalian somatic cell clones frequently exhibit abnormal gene expression that presumably results from errors in reprogramming of the transplanted genome. In the mouse, aggregation of 4-cell stage clones with each other improves reprogramming with respect to Oct-4 expression in blastocysts and an increase in term development (Boiani et al. 2003 EMBO J. 22, 5304-5312). To determine if clone-clone aggregation has a similar beneficial effect in the bovine, we aggregated 8-16 cell bovine clones with each other and profiled gene expression levels in bovine clones and clone-clone aggregates at the blastocyst stage. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39�C in an atmosphere of 5% CO2, 5% O2, and 90% N2. For aggregation of embryos, we first removed the zonae pepellucidae by treatment with 0.5% pronase at the 8-16 cell stage and then placed two zona-free embryos per well into deep microwells produced on the bottom of a culture dish by pressing a heated darning needle onto the surface. Seven to 10 microwells in close proximity were covered by a culture 50-�L drop of culture medium, and embryos were cultured until Day 7. Real-time RT-PCR analysis for Oct-4, DNA methyltransferase 1 (Dnmt1), Dnmt3, glucose transporter 1 (Glut1), Glut3, and Poly(A) polymerase (PolyA) was performed on reusable Dynabead Oligo (dT)25-cDNA libraries synthesized from individual blastocysts at Day 7. In vitro-fertilized embryos were used as controls. To compare the variation of gene expression in each embryo within the group, the coefficient of variation (COV; standard deviation/mean) was calculated. Although spatial distribution of Oct-4 transcript is normal in bovine blastocyst stage clones (Kurosaka et al. 2004 Reprod. Fertil. Dev. 16, 147), we detected disturbances in the level of Oct-4 expression in clones: 44.4% (8 of 18) of clones expressed Oct-4 within a range of 0.5- and 1.5-fold of the average level of expression in IVF embryos, compared to 81.8% (9 of 11) of IVF embryos. Only 22.2% (4 of 18) of clones expressed all genes examined within a range of 0.5- and 2.0-fold of the average level of IVF embryos, versus 45.5% (5 of 11) of IVF embryos. Clone-clone aggregation did not increase the proportion of clones with normal expression levels but did reduce the coefficient of variation of gene expression levels between individual clones for the genes Oct-4, Dnmt1, Dnmt3a and PolyA, but not for Glut1 and Glut3. Interestingly, bovine clone-clone aggregates (n = 25) had less variation between individual embryos compared to IVF aggregates (n = 11) for all genes except Glut1 and Glut3, although variation of single clones was larger than that of single IVF embryos. Analysis of Oct-4 and �-Actin transcripts in mouse clone blastocysts indicated a similar decrease in gene expression variation subsequent to aggregation of mouse clones. These results demonstrate that bovine pre-implantation stage clones exhibit a high degree of variation in gene expression levels and suggest that aggregation of clones is beneficial in reducing the variation in expression of some genes.

194 EXPRESSION OF microRNA-15a, 16, AND 21 AND THEIR POSSIBLE ROLES IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 176
Author(s):  
D. X. Zhang ◽  
X. H. Shen ◽  
X. S. Cui ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Expression Levels ◽  
Apoptotic Gene

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNA molecules that can regulate gene expression by base-pairing with fully or partially sequence-complementary target mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. While miRNAs play an important role in animal development, little is known about their biological function during early mammalian development. In order to obtain insight into the role of miRNAs in early embryogenesis, we first determined the expression levels of three apoptosis-related miRNAs, miR-15a, -16, and -21 in mouse preimplantation embryos using TaqMan� MicroRNA Assays. Five embryos of each developmental stage were snap-frozen and amplified by stem-loop RT primer and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The miRNA concentrations (10–X) in embryo samples were calculated by standard curve from synthetic lin-4 miRNA and the absolute copy number per embryo was obtained based on the formula of 6.02 � 10(8–X). All three miRNAs had low expression levels from the zygote to the 8-cell stage and were up-regulated thereafter. In general, among the three miRNAs, miR-15a exhibited the lowest expression in preimplantation embryos, while miR-16 exhibited the highest. Because of the low levels of miRNA-15a, we determined developmental ability and apoptosis of embryos following microinjection of miRNA-15a. The microinjection of miR-15a into zygotes did not affect embryo development up to the blastocyst stage (miR-15a, 90 � 4.5% v. buffer 94.6 � 5.8%); however, it did induce a significant degree of apoptosis (P < 0.05; Tukey's multiple range test). Furthermore, the expression levels of miR-15a and -16 were increased in microinjected blastocysts compared to the control group (copy number per blastocyst, miR-15a, 6991 � 1223 v. 3098 � 592; miR-16, 196216 � 958 v. 133514 � 6059). Real-time RT-PCR data showed that the gene expression levels of the housekeeping gene GAPDH, the anti-apoptotic gene Bcl-xL, and the miRNA pathway-related genes GW182 and Dicer remained unchanged in miR-15a-injected blastocysts compared to the control group. In contrast, the expression of the stem cell-specific transcriptional factor Oct-4 (fold change, 1.451 � 0.12), the pro-apoptotic gene Bax (1.418 � 0.12), and Caspase 3 (1.314 � 0.19) were significantly increased in microinjected blastocysts. In addition, treatment of 2-cell embryos with 600 µm H2O2 induced apoptosis and increased the expression level of miR-16 at the blastocyst stage (P < 0.05). Taken together, the changes in the expression levels of miR-15a, -16, and -21 in various embryonic developmental stages indicate a possible role for them in early embryogenesis. Furthermore, the high expression levels of miR-15a and miR-16 seem to be linked to apoptosis in blastocyst-stage embryos; this may be due to an increase in the expression of pro-apoptotic genes.

GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues

2005 ◽  
Vol 21 (3) ◽  
pp. 389-395 ◽  
Author(s):  
Robert D. Barber ◽  
Dan W. Harmer ◽  
Robert A. Coleman ◽  
Brian J. Clark
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Gene Expression Data ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Human Tissues ◽  
Expression Data ◽  
Expression Levels ◽  
Gapdh Mrna

Quantitative gene expression data are often normalized to the expression levels of control or so-called “housekeeping” genes. An inherent assumption in the use of housekeeping genes is that expression of the genes remains constant in the cells or tissues under investigation. Although exceptions to this assumption are well documented, housekeeping genes are of value in fully characterized systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. To investigate the value of GAPDH as a housekeeping gene in human tissues, the expression of GAPDH mRNA was measured in a panel of 72 different pathologically normal human tissue types. Measurements were obtained from 371,088 multiplexed, quantitative real-time RT-PCRs with specific target genes. Significant differences in the expression levels of GAPDH mRNA were observed between tissue types and between donors of the same tissue. A 15-fold difference in GAPDH mRNA copy numbers was observed between the highest and lowest expressing tissue types, skeletal muscle and breast, respectively. No specific effect of either age or gender was observed on GAPDH mRNA expression. These data provide an extensive analysis of GAPDH mRNA expression in human tissues and confirm previous reports of the marked variability of GAPDH expression between tissue types. These data establish comparative levels of expression and can be used to add value to gene expression data in which GAPDH is used as the internal control.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
A. Baji Gal ◽  
S. Mamo ◽  
S. Bodo ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Standard Error ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Mouse Embryos ◽  
Cell Stage ◽  
The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P &lt; 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

265 DICER GENE EXPRESSION IN PRE-IMPLANTATION MOUSE EMBRYOS: IMPLICATION OF TRANSCRIPTION REGULATION AT THE BLASTOCYST STAGE

2007 ◽  
Vol 19 (1) ◽  
pp. 249
Author(s):  
X. S. Cui ◽  
X. H. Shen ◽  
X. Y. Li ◽  
J. M. Kim ◽  
N. H. Kim
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Transcription Factors ◽  
Real Time ◽  
Expression Profiles ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Expression Levels ◽  
Mouse Oocytes ◽  
Stage Embryo

Dicer is an RNAse III enzyme that is related to the generation of the microRNAs involved in the gene silencing pathway. In order to obtain insight into the role of Dicer in early embryo development, we first evaluated its gene expression levels in mouse oocytes and embryos during in vitro development. The relative abundance of Dicer1 transcripts was established by real-time RT-PCR using the 2-ddCt method. H2a was applied as an internal standard to normalize the real-time RT-PCR reaction efficiency and quantify Dicer1 mRNA. Relatively high expression levels of mRNA in germinal vesicle-stage oocytes steadily decreased up to the 2-cell stage embryo, and then expression remained during morulae and blastocyst formation. Protein synthesis of Dicer was also observed in the mouse oocytes and early embryos. Specific silencing of mRNA expression and protein synthesis by RNA interference (siRNA) did not inhibit developmental events up to the blastocyst (BL) stage. However, Dicer1 siRNA reduced (P &lt;0.05) total nuclei numbers in the BL-stage embryos (Dicer1: 77.2�4.2 vs. control: 62.7�3.1). Real-time RT-PCR also confirmed that, following Dicer1 siRNA microinjection into zygotes, transcription levels of several non-target genes, Cdc42, Cdh1, Dbc2, ILK, Tuba1, Plat, and Tie1, were not changed in blastocyst-stage embryos. However, selected transcription factors, Pou5f1 (P &lt;0.01), Nanog (P &lt;0.005), and Sox2 (P &lt;0.01), in blastocysts were significantly down-regulated. Additionally, POU5F1 protein synthesis was also reduced. Using Applied Biosystem microarray technology, we compared gene expression profiles in control and Dicer1 siRNA microinjected blastocysts. This technique confirmed that 397 or 737 of 16354 genes were up- or down-regulated, respectively, following siRNA microinjection (P &lt;0.05), including 52 transcription factors. The results suggest that expression of Dicer regulates gene expression at the blastocyst-stage embryo for cell fate, possibly by the transcription control.

Themicrorna-146boverexpression Helps To Differentiate Benign Nodules FromPapillary Thyroid Carcinoma.

2018 ◽  
Vol 9 (SPL1) ◽  
Author(s):  
Shoroq Mohammed AL Temimi ◽  
Adel Mosa AL-Rekabi
Keyword(s):  
Gene Expression ◽  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Characteristic Curve ◽  
Housekeeping Gene ◽  
Papillary Thyroid ◽  
Stem Loop ◽  
Expression Levels

MicroRNA-146b is one of the widely studied microRNAs in thyroid cancers and has been shown to be frequently upregulated in papillary thyroid carcinoma and plays a crucial role in tumorigenesis, progression and may become a potential therapeutic target and biomarker of tumor diagnosis and prognosis. Estimation of microRNA-146b gene expression levels in fresh tissues of solitary thyroid nodulein comparison to normal adjacent tissue of same patients by using stem-loop follow by Taq-Man Real Time PCR (RT-PCR) technique to distinguish the benign nodule from papillary carcinoma and correlate with age, genderof patients,size of tumor,lymph node involvement and stage of tumors. Stem-loop Real time -PCR was performed to identify the level of microRNA-146b gene expression in fresh tissues of solitary thyroid nodule in comparison to normal adjacent tissues of same patients. The expression levels of microRNA-146b were normalized to housekeeping gene by using the livak method. The expression level of tissue microRNA-146b in papillary thyroid carcinoma cases was significantly higher than that benign cases and normal adjacent tissues. Receiver operating characteristic curve analyses indicated that use of tissue microRNA-146b have a high diagnostic sensitivity and specificity for papillary thyroid carcinomain correlated with nodal status and advance tumor- stage. The microRNA‑146b as a new novel diagnostic and prognostic biomarker forpapillary thyroid carcinoma.

scholarly journals Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Ren Chen ◽  
Mayumi Gyokusen ◽  
Yoshihisa Nakazawa ◽  
Koichiro Gyokusen
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Transgene Expression ◽  
Pcr Amplification ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Amplification Efficiency ◽  
Eucommia Ulmoides ◽  
Rt Pcr

In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT-) PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT-) PCR amplification efficiency, gene expression stability, and transgene influences. We used the strategy to select two genes, ACTα and EF1α, from 10 candidate housekeeping genes, as the optimal housekeeping genes to evaluate transgenic Eucommia ulmoides Oliver root lines overexpressing IPPI or FPPS1 genes, which are involved in isoprenoid biosynthesis.

Validation of Stable Housekeeping Genes for Quantitative Real Time PCR in Golden Syrian Hamster

2020 ◽  
Author(s):  
Jin-xin Miao ◽  
Jian-yao Wang ◽  
Nick Robl ◽  
Hao-ran Guo ◽  
Shao-he Song ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Real Time ◽  
Syrian Hamster ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Pancreatic Tissue ◽  
Golden Syrian Hamster ◽  
The Stability ◽  
Selection Of

Background: Golden Syrian hamster (GSH) have many advantages as animal models in preclinical research, but their application is currently limited by the lack of standardized techniques for analyzing gene expression. Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for analyzing gene expression, but its reliability depends upon the selection of stable reference (“housekeeping”) genes for proper normalization. The current study was aimed to RT-qPCR for investigated to determine the stability housekeeping gene in golden Syrian hamster.Methods: During the period of June 2019 to October 2019, the expression stability of eight commonly-used housekeeping genes (glyceraldehyde-3phosphate dehydrogenase, Gapdh; b-actin, Actb; hypoxanthine phosphoribosyl transferase 1, Hprt1; ribosomal protein L13a, Rpl13a; ribosomal protein S18, Rps18; Beta-2-microglobulin, B2m; Tubulin beta class I, Tubb; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta, Ywhaz) were investigated in lung, liver, spleen and pancreatic tissue of golden Syrian hamsters using BestKeeper, geNorm, NormFinder software, comparative delta Ct method and RefFinder. Result: It was found that the stability of gene expression varied among tissue where in Actb was the most stably expressed housekeeping gene for lung tissue, Hprt1 for liver, Rpl13a for spleen and Tubb for pancreatic tissue. In contrast, the least stable housekeeping gene in both liver and spleen was Gapdh, Rps18 in pancreas and Ywhaz in lung tissue. These data provide a critical evaluation of housekeeping genes in GSH tissues that can be used as a guide for selection of the appropriate genes in future studies.

scholarly journals Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kim Hoa Ho ◽  
Annarita Patrizi
Keyword(s):  
Gene Expression ◽  
Choroid Plexus ◽  
Sensory Deprivation ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Experimental Conditions ◽  
Brain Repair ◽  
Expression Studies ◽  
Testing Algorithms ◽  
Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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