RNA degradation differentially affects quantitative mRNA measurements of endogenous reference genes in human placenta

It has been highlighted that RNA quality and appropriate reference gene selection is crucial for the interpretation of RT-qPCR results in human placental samples. In this context we investigated the effect of RNA degradation on the mRNA abundance of seven frequently used reference genes in 119 human placental samples. Combining RNA integrity measurements, RT-qPCR analysis and mathematical modeling we found major differences regarding the effect of RNA degradation on the measured expression levels between the different reference genes. Furthermore, we demonstrated that a modified RNA extraction method significantly improved RNA quality and consequently increased transcript levels of all reference genes.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

mRNA expression and binding sites for alpha2-adrenergic receptor subtypes in muscle layers of the ileum and spiral colon of dairy cows

OBJECTIVE: To measure maximum binding capacity (B(max)) and levels of mRNA expression for alpha(2)-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows. SAMPLE POPULATION: Ileal and colonic muscle specimens from 6 freshly slaughtered cows. PROCEDURES: Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for alpha(2)-AR subtypes was measured by real-time reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 ((3)H-RX821002) and subtype-selective ligands as competitors. RESULTS: mRNA expression for alpha(2AD)-, alpha(2B)-, and alpha(2C)-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for alpha(2AD)-AR was significantly greater than that for alpha(2B)- and alpha(2C)-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of (3)H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The B(max) of alpha(2AD)- and alpha(2C)-AR sub-types was greater than that of alpha(2B)-AR. The B(max) and level of mRNA expression were only correlated (r = 0.8) for alpha(2AD)-AR. Ratio of B(max) to mRNA expression for alpha(2C)-AR was similar to that for alpha(2B)-AR, but significantly greater than for alpha(2AD)-AR. CONCLUSIONS AND CLINICAL RELEVANCE: Subtypes of alpha(2)-AR in bovine intestinal muscle layers are represented by a mixture of alpha(2AD)- and alpha(2C)-ARs and of alpha(2B)-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in alpha(2)-adrenergic mechanisms regulating bovine intestinal motility.

Quantitative mRNA analysis of adrenergic receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD). SAMPLE POPULATION: Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter). PROCEDURES: Concentrations of mRNA for 9 adrenoceptor subtypes (alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), beta(1), beta(2), and beta(3)) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA for alpha(1B)-, alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors, and in the cecum and PLAC for alpha(2B)- and beta(2)-adrenoceptors. Groups did not differ significantly for alpha(1A)-adrenoceptors. The mRNA expression for alpha(1D)-, alpha(2C)-, and beta(3)-adrenoceptors was extremely low in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Distribution of mRNA coding for 5-hydroxytryptamine receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 7 subtypes of 5-hydroxytryptamine receptors (5-HTRs) in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation (CDD). SAMPLE POPULATION: Full-thickness intestinal wall biopsy specimens were obtained from the ileum, cecum, proximal loop of the ascending colon, and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy dairy cows allocated to 2 control groups (specimens collected during routine laparotomy [group 2] or after cows were slaughtered [group 3]). PROCEDURE: Amounts of mRNA coding for 7 subtypes of 5-HTRs (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT4) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed as the percentage of mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA coding for 5-HTR1B, 5-HTR2B, and 5-HTR4 was significantly lower in cows with CDD than in healthy cows. For 5-HTR2B and 5-HTR4, significant differences between cows with CDD and control cows were most pronounced for the ELSC. Expression of mRNA for 5-HTR1D, 5-HTR1F, and 5-HTR2A was extremely low in all groups, and mRNA for 5-HTR1A was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Relative concentrations of mRNA coding for 5-HTR1B, 5-HT2B, and 5-HTR4 were significantly lower in the intestines of cows with CDD than in the intestines of healthy dairy cows, especially for 5-HT2B and 5-HTR4 in the ELSC. This supports the hypothesis that serotonergic mechanisms, primarily in the spiral colon, are implicated in the pathogenesis of CDD.

Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying

It has been proposed that gonadotropins and/or gonadotropin releasing hormone (GnRH) could be involved in the pathophysiology of the side effects after spaying in bitches, such as urinary incontinence and an increased production of a woolly undercoat. In order to provide tools to investigate the role of these hormones in dogs we developed immunohistochemical techniques and real-time RT-PCR to study whether GnRH-, LH-, and FSH-receptors exist in canine skin and urinary bladder. Tissue samples from the skin of the flank region and the ventral midline of the urinary bladder from euthanised dogs were examined. We were able to quantify mRNA expression of GnRH-, FSH-, and LH-receptors in canine skin and bladder biopsies with a high primer efficacy. Immunohistochemical studies showed that GnRH-, FSH-, and LH-receptors are expressed in vessel walls, the epidermis, the hair follicle and in sebaceous and sweat glands in canine skin and in transitional epithelium, and smooth muscle tissue in the urinary bladder. Our data provide the fundamentals to examine the distribution of FSH-, LH-, and GnRH-receptors in canine skin and urinary bladder and to assess gene activity at the transcriptional level by real-time RT-PCR.

Ontogenesis of mRNA levels and binding sites of hepatic alpha-adrenoceptors in young cattle

Catecholamines affect hepatic glucose production through (alpha- and beta2-) adrenoceptors (AR). We studied mRNA abundance and binding of hepatic alpha-AR in pre-term (P0) calves and in full-term calves at day 0 (F0), day 5 (F5) and day 159 (F159) to test the hypothesis that gene expression and numbers of hepatic alpha-AR in calves are influenced by age and associated with beta2-AR and selected traits of glucose metabolism. mRNA levels of alpha1- and alpha2-AR were measured by real time RT-PCR. alpha1- and alpha2-AR numbers (maximal binding, Bmax) were determined by saturation binding of (3H)-prazosin and (3H)-RX821002, respectively. alpha1- and alpha2-AR subtypes were evaluated by competitive binding. alpha1A-AR mRNA levels were lower in P0 than in F0, F5 and F159 and alpha(2AD)-AR mRNA levels were lower in F159 than in P0, F0 and F5, while alpha2C-AR mRNA levels increased from P0 and F0 to F5 and F159. Bmax of alpha1-AR increased from P0 to F5, then decreased in F159. Bmax of alpha2-AR decreased from F0 to F159. Bmax of alpha1-AR was positively associated with mRNA levels of alpha1A-AR (r = 0.7), Bmax of beta2-AR (r = 0.5) and negatively with hepatic glycogen content (r = -0.6). Bmax of alpha2-AR was negatively associated with Bmax of beta2-AR (r = -0.4). In conclusion, mRNA levels and binding sites of alpha1- and alpha2-AR in calves exhibited developmental changes and were negatively associated with hepatic glycogen content.