Rna fix

RNA was isolated from three different sections of the oviduct tube (INF, DM, and PM) and cultivated oviduct cells, derived from the respective birds. For in vivo assay, INF, DM, and PM fragments, each 1 cm long, were cut off aseptically and put separately into Eppendorf tubes containing 3.0 mL RNAfix (EURx, Gdansk, Poland). Tissue samples were kept for 24 h at 4 °C and subsequently stored at − 20 °C until isolation of RNA. For RNA isolation from COEC, confluent cells were detached using Accutase® solution (A&E Life Sciences, Gentaur, Sopot, Poland) and centrifuged at 220×g for 5 min at room temperature (RT). Cell pellets were resuspended in 0.5 mL RNAfix (EURx, Gdansk, Poland) to preserve cells prior to RNA isolation. RNA was extracted using the universal RNA purification kit (EURx, Gdansk, Poland) according to manufacturer’s recommendation. RNA was quantified using spectrophotometry and RNA quality by gel electrophoresis.

Total DNA was isolated from trigeminal ganglia and brains preserved in RNA fix (Eurx) using RNA/DNA Extracol kit (Eurx), according to the manufacturer’s instructions. HSV-1 was detected using an HSV-1 probe labeled with FAM (carboxyfluorescein) in quantitative polymerase chain reaction (qPCR) instrument ViiA 7 (Fast block; Applied Biosystems) with Fast Advanced Master Mix (Thermo Fisher Scientific) as described by Namvar et al [19 (link)] and Cymerys et al [20 (link)].

The cecal mucosa (n = 5 per group) was collected for gene expression analysis after slaughter. The cecum from each individual was cut lengthways after collection and rinsed in PBS. The mucosal layer was scraped with a glass slide. The collected tissues were fixed in RNA stabilizing buffer (RNA fix; EURx, Gdansk, Poland). Each tissue was homogenized in 1 mL of TRIzol reagent (MRC, Cincinnati, OH, USA) by using a TissueRuptor homogenizer (Qiagen GmbH, Hilden, Germany). 200 μL of chloroform was added to the homogenate, shaken, and centrifuged (12,000 rpm, 15 min). The aqueous phase with the isolated RNA was collected. RNA was additionally purified using a commercial set- Universal RNA Purification Kit (EURx, Gdansk, Poland) according to the manufacturer’s instructions. RNA was eluted in a volume of 50 μL of nuclease-free water. Qualitative and quantitative control of RNA was performed using a 2% agarose gel electrophoresis and spectrophotometer (Nanodrop 2000; Thermo Scientific, Wilmington, NC, USA). The RNA was stored at −20 °C, as recommended by the manufacturer of the isolation set.