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PubMed2005

Stanniocalcin in the euryhaline flounder (Platichthys flesus): primary structure, tissue distribution, and response to altered salinity.

Stanniocalcin (STC) is a homodimeric glycoprotein hormone that was first discovered in fish, where it is largely produced by a unique endocrine gland, the corpuscles of Stannius (CS). In bony fish, it is thought to be an important regulator of calcium and phosphate uptake from the aquatic environment. This report describes the molecular cloning of STC from euryhaline flounder (Platichthys flesus) CS cDNA and genomic DNA. The flounder STC encodes a prehormone of 251 amino acids (aa) with a signal peptide of 17 aa, followed by another 15 aa sequence before the mature protein of 219 aa. The deduced aa sequence of flounder STC shows 62.9-89.0% similarity and 50.4-83.1% identity with other known fish STC sequences, but only 42.3% identity with mouse STC1, 24.4% identity with fugu and zebrafish STC2, and 22.3% identity with mouse STC2. Primary structural analysis demonstrated that flounder STC gene contains five exons in contrast to the four exons present in mammalian STC gene. RT-PCR revealed the expression of flounder STC mRNA to be widely spread in many tissues and organs, similar to the situation in mammals and other fish. Quantitative PCR (Q-PCR) was conducted to measure relative STC expression levels in the CS, which showed STC mRNA expression levels in seawater-adapted fish CS were about 3-fold higher than in freshwater-adapted fish CS.

Adaptation, Physiological↗
PubMed2004

Changes in aquaporin expression in the inner ear of the rat after i.p. injection of steroids.

The aquaporins (AQPs) are a family of small transmembrane water transporters. It has recently been revealed that they play a role in regulating homeostasis in the inner ear fluids. Steroid therapy is usually administered to patients with inner ear disorders; however, the mechanism of steroid effects has not been clearly determined. To elucidate the points of action of steroids in the inner ear, we recently examined the distributions of AQP isoform mRNAs in the rat inner ear and identified AQP1-6 mRNAs in the rat cochlea and AQP1, 3, 4, 5 and 6 mRNAs in the rat endolymphatic sac by means of reverse transcriptase polymerase chain reaction (PCR). In this study, we investigated changes in expression of AQP mRNAs in the rat inner ear after i.p. injections of steroids using real-time quantitative PCR and found that AQP3 mRNA in the endolymphatic sac was significantly upregulated in both dose- and time-dependent manners. This result suggests that steroids may effect water homeostasis in the rat inner ear via AQPs.

Animals↗
PubMed2004

Stratification of activity and bacterial community structure in biofilms grown on membranes transferring oxygen.

Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s(-1)) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s(-1), AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s(-1), AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.

Bacteria↗
PubMed2004

Psoriasin is aberrantly expressed in human breast cancer and is related to clinical outcomes.

Psoriasin (S100A7) is a member of the S100 gene family that is involved in psoriasis, which can be induced in cultured squamous epithelial cells. Previously, this molecule has been shown to be over-expressed in human ductal carcinoma in situ and invasive breast cancers. This current study examine the levels of expression of this molecule in a group of human breast tumours, with particular emphasis on the relationship with the clinical outcomes using quantitative PCR and the distribution in tumours using immunohistochemistry. Psoriasin is primarily expressed in breast cancer cells, and at very low level in normal breast epithelial cells. Quantitative analysis of psoriasin mRNA has shown that breast tumour tissues exhibited a significantly high level of psoriasin compared with normal tissues (p=0.0026). The levels do not correlate with nodal status of breast tumours, however levels in grade 2 and grade 3 tumours were significantly higher compared with that in grade 1 tumours (p=0.07 and p=0.0015, vs grade 1 respectively). Lobular carcinoma also had higher levels of psoriasin compared with ductal tumours. The most interesting observation is that levels of psoriasin were significantly higher in patients who developed metastatic disease and in patients who died of breast cancer (p=0.02 and p<0.001, vs disease-free, respectively). It is concluded that aberrant expression of psoriasin is commonly seen in human breast cancer and that excessively high levels are correlated with the clinical outcomes.

Biomarkers, Tumor↗
PubMed2005

Expression of the epidermal growth factor seven-transmembrane member CD97 correlates with grading and staging in human oral squamous cell carcinomas.

INTRODUCTION: The oral squamous cell carcinoma (OSCC) is the sixth most common malignant tumor worldwide. No significant better progress has been made in the treatment of OSCCs during the last decades. The heterodimeric CD97 protein is a epidermal growth factor seven-transmembrane family member and was identified as a dedifferentiation marker in thyroid carcinomas. Nothing is known about CD97 in OSCCs. MATERIAL AND METHODS: Employing UV-laser microdissection, CD97 and its ligand CD55 were investigated in normal oral mucosa and OSCCs (n = 78) by multiplex reverse transcription-PCR. Frozen sections were investigated by immunohistochemistry. The effects of retinoic acid and sodium butyrate on the CD97/CD55 expression in OSCC cell lines were determined by quantitative PCR, immunocytochemistry, and flow cytometry. RESULTS: Weak CD97 transcripts were expressed in normal mucosa and normal basal epithelial cells revealed specific CD97 immunostaining. Strong CD97 transcripts were detected in pT(3)/T(4) and G3/G4 OSCC tissues, whereas pT(1)/T(2) and G1/G2 carcinomas revealed weak CD97 transcript levels. A weak CD97 immunostaining was observed in pT(1)/T(2) and G1/G2 tumors. By contrast, intensive CD97 immunostaining was detected in pT(3)/T(4) OSCCs and G3/G4 lesions. CD55 gene expression was low in normal mucosa. All OSCCs, irrespective of stage and grading, displayed strong CD55 immunostaining. Sodium butyrate and retinoic acid inhibited CD97 mRNA and protein in OSCC cell lines. Interestingly, CD55 was up-regulated by both substances. CONCLUSION: We identified CD97 as a novel marker of dedifferentiated OSCC. Interaction of CD97 and CD55 may facilitate adhesion of OSCC cells to surrounding surfaces that would result in metastases and bad prognosis.

Adult↗
PubMed2005

[Diagnostic difficulties of Toxoplasma gondii infection in pregnant women. Is it possible to explain doubts by polymerase chain reaction?].

OBJECTIVES: Toxoplasma gondii infection during pregnancy is still a difficult problem in the contemporary perinatology. Difficulties met during interpretation of serological tests carried out in pregnant patients to detect Toxoplasmosis implies more and more frequent use of the Polymerase Chain Reaction (PCR). DESIGN: To evaluate the dependence between serological tests and quantity of the Toxoplasma gondii genomes in mothers' blood and amniotic fluid or neonatal blood, the quantitative PCR (q-PCR) method was applied. MATERIALS AND METHODS: The analysis was performed in 81 pregnant women. Maternal blood, amniotic fluid and newborns' umbilical blood samples were evaluated for the presence of Toxoplasma gondii DNA. IgG and IgM Toxoplasma gondii antibodies were evaluated by the ELISA method. RESULTS: High seroprevalence (51.9%) of the Toxoplasma gondii was confirmed. Toxoplasma gondii genetic material in blood and/or amniotic fluid was found in 33 patients. It was stated that quantity of the protozoa and anti-IgM presence in mothers' blood are the factors influencing significantly the Toxoplasma gondii manifestation in amniotic fluid. CONCLUSION: High suitability of PCR in diagnosis of Toxoplasmosis during pregnancy and vertical transmission was confirmed.

Adult↗
PubMed2006

High variability of human cytomegalovirus UL150 open reading frame in low-passaged clinical isolates.

OBJECTIVE: To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection. METHODS: PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplification products were sequenced directly, and the data were analyzed. RESULTS: Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMV UL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0.1% to 6.8% and the amino acid diversity was 0.2% to 19.2% related to Toledo strain. However, the nucleotide diversity was 0.1% to 6.4% and amino acid diversity was 0.2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo. CONCLUSION: HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.

Amino Acid Sequence↗
PubMed1993

Assessment of G-CSF and GM-CSF mRNA expression in peripheral blood mononuclear cells from patients with severe congenital neutropenia and in human myeloid leukemic cell lines.

Patients with severe congenital neutropenia (SCN), also called Kostmann syndrome, are unable to generate sufficient peripheral blood granulocytes owing to an arrest of myeloid differentiation at the level of promyelocytes. Similarly, myeloid leukemic cells show a maturation arrest at different stages of myeloid maturation coupled with uncontrolled proliferation. Among other cells, defective production of or defective response to granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) might be involved in the pathophysiology of these disorders of hematopoiesis. Reverse transcription of messenger RNA and subsequent specific amplification by the polymerase chain reaction (RT-PCR) served as a sensitive technique to detect G-CSF and GM-CSF gene expression. We have tested two alternative assays for the specific quantitation of transcript levels for G-CSF. Applying one assay we could demonstrate that: 1) peripheral blood monocytes from 5 patients with SCN are able to express G-CSF and GM-CSF messenger RNA, suggesting that defective production of these factors is not responsible for the neutropenia in this condition; 2) messenger RNA levels from 5 SCN patients were on average higher than the levels determined for three healthy volunteers; 3) 7 of 9 of the examined myeloid cell lines express GM-CSF and all of them G-CSF mRNA. These results show that quantitative PCR techniques can be used as simple tools to elucidate aspects of the pathophysiology of hematologic disorders concerning the production of CSFs.

Adult↗
PubMed1997

Interleukin (IL)-1 beta and IL-1 beta mRNA expression in normal and diseased skeletal muscle assessed by immunocytochemistry, immunoblotting and reverse transcriptase-nested polymerase chain reaction.

To confirm the production of IL-1 beta and to optimize detection and semiquantitation of IL-1 beta mRNA by polymerase chain reaction (PCR) techniques in skeletal muscle tissue, immunocytochemistry, immunoblotting and several procedures of RNA extraction and reverse transcription (RT)-PCR amplification were used on muscle samples from 12 patients with conditions associated with local production of IL-1 beta (AZT myopathy: 6 patients; sarcoid myopathy: 6 patients) and from 9 patients with normal muscle used as controls. Abundant IL-1 beta immunoreactivities, corresponding to both pro IL-1 beta and mature IL-1 beta as assessed by immunoblotting, were observed in all diseased muscles, either in inflammatory cells (sarcoid myopathy) or in atrophic muscle fibers (AZT myopathy). Acid guanidinium isothiocyanate phenol-chloroform extraction of RNA appeared less efficient for IL-1 beta mRNA detection by RT-PCR than proteinase K digestion followed by phenol-chloroform extraction. Even using the latter procedure, RT-single PCR for IL-1 beta mRNA was puzzlingly negative in all cases but one; in contrast, RT-nested PCR specified by DNA enzyme immunoassay yielded detection of IL-1 beta mRNA in all diseased muscles and in occasional controls, including the expected PCR product of 391 bp, but also another product of 935 bp, corresponding to IL-1 beta mRNA with unsplicing of the fourth intron. Semi-quantitative PCR showed that production of IL-1 beta mRNA was higher in sarcoid myopathy than in AZT myopathy, and in AZT myopathy than in controls. In conclusion, IL-1 beta expression can be reliably studied using immunocytochemistry, but assessment of IL-1 beta mRNA production in muscle tissue requires optimized extraction and RT-PCR procedures.

Atrophy↗
PubMed2025

Development of a plant-based vaccine against brucellosis: stable expression of Brucella abortus OMP25 in transgenic tobacco.

Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.

Plants, Genetically Modified↗
PubMed2003

hTERT gene amplification and increased mRNA expression in central nervous system embryonal tumors.

High-level gains at 5p15, a chromosomal region including the human telomerase catalytic protein subunit (hTERT) gene, have been documented in several medulloblastomas. We therefore analyzed hTERT gene dosage in a group of medulloblastomas and other embryonal brain tumors using differential PCR. Amplification of the hTERT locus was detected in 15 of 36 (42%) tumors examined. To correlate gene amplification with message level, we used real-time quantitative PCR to measure hTERT mRNA in 50 embryonal brain tumors. hTERT mRNA was detected in all but one of these cases, and mRNA level correlated significantly with gene dosage (r = 0.82). Log-rank analysis of survival data revealed a trend toward poor clinical outcomes in patients with medulloblastomas containing high hTERT mRNA levels, but clinical follow-up was relatively short and the association was not statistically significant (P = 0.078). Comparative genomic hybridization was used to further analyze the tumor with the greatest hTERT gene dosage and mRNA level, a recurrent medulloepithelioma. hTERT was amplified in the recurrent tumor but not in the primary lesion, suggesting this locus can be involved in tumor progression. Our data indicate that hTERT gene amplification is relatively common in embryonal brain tumors, and that increased expression of hTERT mRNA may be associated with biologically aggressive tumor behavior.

Adolescent↗
PubMed2000

A simple method for the rapid generation of recombinant adenovirus vectors.

Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.

Adenoviridae↗
PubMed2000

Activation of metabotropic GABAB receptors inhibited glutamate responses in spiral ganglion neurons of mice.

The lateral olivocochlear efferent (LOC) fibers form axodendritic synapses with auditory nerve dendrites synapsing on inner hair cells to affect postsynaptic responses GABA is one of the primary transmitters used by the LOC efferent. However, the molecular nature and developmental expression of the GABA receptors in spiral ganglion (SG) neurons are poorly understood. We have identified the GABAB receptors in SG neurons of mice by pharmacological criteria and polymerase chain reaction (PCR) detection. Applications of GABA or baclofen increased intracellular Ca2+ concentrations and inhibited glutamate responses. The responses were blocked by saclofen, but not by bicuculline. Relative-quantitative PCR showed that GABAB receptors were expressed prenatally, and their levels did not change significantly during the maturation of SG neurons.

Age Factors↗
PubMed2006

Identification and characterization of an antigen I/II family protein produced by group A Streptococcus.

Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.

Alleles↗
PubMed2006

Disruption of the gene encoding the beta1-subunit of transducin in the Rd4/+ mouse.

PURPOSE: The Rd4/+ mouse inherits an autosomal dominant retinal degeneration that cosegregates with a large inversion spanning nearly all of mouse chromosome 4 (Chr 4). This inversion is homozygous lethal. The hypothesis for the study was that disruption of a gene at one of the two breakpoints in the Rd4 chromosome is responsible for the retinal degeneration. The purpose was to identify the disrupted gene. METHODS: Genotyping was performed by PCR and gel electrophoresis. The Rd4/+ phenotype was confirmed by ERG. Fluorescence in situ hybridization (FISH) analysis was performed with bacterial artificial chromosome (BAC) probes. Northern and quantitative PCR procedures were used to evaluate Gnb1 mRNA expression. Protein expression was measured by Western blot. RESULTS: To identify the Rd4 gene defect, the breakpoints were first localized with a testcross and the locus refined by using FISH. Genetic testcross data revealed that the inversion breakpoints are located within a few centimorgans of both the telomeric and centromeric ends of Chr 4. Initial FISH analysis showed the proximal breakpoint of the inversion to be in the centromere itself. Therefore, we focused on the distal breakpoint and found that it lies in the second intron of the gene Gnb1, coding for the transducin beta1-subunit (Tbeta1) protein that is directly involved in the response to light of rod photoreceptors. Before the beginning of retinal degeneration in Rd4/+ retina, the levels of Gnb1 mRNA and Tbeta1 protein are 50% of those in wild-type retina. CONCLUSIONS: The results suggest that disruption of the Gnb1 gene is responsible for Rd4 retinal disease.

Animals↗
PubMed2002

Evidence for hepatocyte differentiation from embryonic stem cells in vitro.

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.

Albumins↗
PubMed2001

Application of the 5' fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA analysis in sediments.

In this study, we report on the development of quantitative PCR and reverse transcriptase PCR assays for the 16S rRNA of Geobacter spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. The lower detection limit of each assay was 5 to 50 fg of genomic DNA or < or =2 pg of 16S rRNA. TaqMan PCR spectral traces from uncontaminated, amended aquifer sediments were significantly lower (P < 0.0002) than traces for the external standard curve. We also observed a similar, significant decrease in mean quencher emissions for undiluted extracts relative to those for diluted extracts (P < 0.0001). If PCR enumerations were based solely upon the undiluted sample eluant, the TaqMan assay generated an inaccurate result even though the threshold cycle (C(t)) measurements were precise and reproducible in the sediment extracts. Assay accuracy was significantly improved by employing a system of replicate dilutions and replicate analyses for both DNA and rRNA quantitation. Our results clearly demonstrate that fluorescence quenching and autofluorescence can significantly affect TaqMan PCR enumeration accuracy, with subsequent implications for the design and implementation of TaqMan PCR to sediments and related environmental samples.

DNA, Ribosomal↗
PubMed2005

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.

BACKGROUND: Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the successful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. RESULTS: In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. CONCLUSION: Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

Animals↗