Digoxigenin-labelled probes were prepared using the DIG-RNA-labelling mix (Roche) according to the manufacturers protocol

Digoxigenin-labelled probes were prepared using the DIG-RNA-labelling mix (Roche) according to the manufacturers protocol. observed neuronal processes with angiotensinergic synapses en ATR-101 passant, colocalized with synaptophysin, within the DRG. In the DRG, we also recognized by qRT-PCR, manifestation of Ang II receptor AT1A and AT2-mRNA ATR-101 while AT1B-mRNA was not traceable. In some neurons Compound P and CGRP were found colocalized with Ang II. The intracellular localization and Rabbit polyclonal to MAP1LC3A colocalization of Ang II with Compound P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the rules of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human being DRG and act as a neurotransmitter. hybridization experiments. For extraction of total RNA, rats were soon anesthetized with halothane and consequently sacrificed by decapitation. For extraction of total RNA, new rat thoracic DRG, liver, lung, adrenal glands and kidneys were dissected and instantly transferred into RNA later on (Ambion), freezing in liquid nitrogen, and then processed for total RNA extraction (Ambion). For specific measurement of different angiotensin peptides separated by high performance liquid chromatography (HPLC) prior to highly sensitive radioimmunoassay, rat thoracic DRG were rapidly eliminated, rinsed with chilly Ringer remedy, blotted by filter paper and wet weight was measured. The ganglia were freezing in liquid nitrogen and stored at ?70 C. Human being thoracic DRG were procured from adult human being individuals for whom ATR-101 a permit for medical autopsy (educated written consent by next of kin) had been acquired according to state law, in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Human being DRG were fixed by immersion in freshly prepared 2% formaldehyde for 3 days and then utilized for cryosectioning or inlayed in paraffin. To perform HPLC-RIA the same method as explained for rats was used. 2.2. RNA isolation and quantitative real time PCR (qRT-PCR) New rat DRG were dissected as mentioned above and instantly transferred into RNA later on (Ambion), freezing in liquid nitrogen, and then processed for total RNA extraction (Ambion). RNA integrity was confirmed for each sample within the Agilent Bioanalyzer using the RNA 6000 Nano kit (Agilent Systems). 1 g of total RNA was reverse transcribed using ATR-101 Superscript II (Invitrogen) and random hexamers according to the manufacturers protocol. For qRT-PCR, reverse transcribed material corresponding to 40 ng RNA was amplified with the TaqMan assays explained below in 25 l Common PCR Master Blend, No AmpErase UNG within the SDS 7000 (Applied Biosystems) using the standard thermal protocol. Average ideals and standard deviations of relative mRNA levels of each sample, normalized to relative 18S rRNA levels, are from four measurements and were determined using the relative differences. Ideals were indicated as percent of ideals from liver for Ang-N and cathepsin D, kidney for renin, lung for ACE, and adrenal gland for angiotensin receptors. The following TaqMan assays were utilized for qRT-PCR at a final concentration of 250 nM TaqMan probe and 900 nM of each primer: Angiotensinogen Forward primer 5-CACGACTTCCTGACTTGGATAAAGA-3 Reverse primer 5-CTGCGGCAGGGTCAGA-3 TaqMan probe 5-FAM CCTCGGGCCATCC G MGB-3 manufactured as Assays-by-Design (RATG-EJ3) by Applied Biosystems Renin Assay-on-demand Rn00561847_m1 from Applied Biosystems ACE Assay-on-demand Rn00561094_m1 from Applied Biosystems Cathepsin D Assay-on-demand Rn00592528_m1 from Applied Biosystems AT1A Assay-on-demand Rn01435427_m1 from Applied Biosystems AT1B Assay-on-demand Rn02132799_s1 from Applied Biosystems AT2 Forward primer 5-GTGGGAAGCTCAGTAAGCTGATTTA-3 Reverse primer 5-GTCAGAGACTCCCAATCCTTACAC-3 TaqMan probe 5-FAM ACACTGGCAACTAAAAGA MGB-3 manufactured as Assays-by-Design (RATG-EJ3) by Applied Biosystems 18S rRNA Predeveloped Assay Reagent 431-9413E from Applied Biosystems 2.3. In situ hybridization 2.3.1. DIG-labelled RNA probe preparation By using an appropriate cDNA template for Ang-N [35], a 403 bp long fragment related to nucleotides 221C623 was generated by digestion with restriction enzymes and and into.