Nuclease free distilled water

PAGE/HPLC-purified RNA and DNA oligonucleotides were reconstituted to 100 μM with nuclease-free distilled water (Invitrogen). RNA:DNA hybrids were formed using 20 μM of each complementary oligonucleotide denatured in 60 mM KCl, 50 mM Tris (pH 8.0) at 95°C for 5 min followed by gradual cooling to room temperature. Resulting RNA:DNA duplexes were concentrated by precipitation with 2.5 volumes of 100% ethanol, 0.3 M sodium acetate (pH 5.2) and resuspended in an appropriate volume of nuclease-free TE (10 mM Tris, 0.1 mM EDTA). Purification was performed on an ÄKTA FPLC™ machine (GE Healthcare) at 4°C by injecting 100 μl volume of nucleic acids onto a 24-ml Superdex 10/300 GL column (GE Healthcare) pre-equilibrated with two column volumes of nuclease-free TE. Elution was performed with equilibration buffer (TE) at a flow rate of 0.4 ml/min, with 100 μl fractions collected into sterile nuclease-free 96-well 2-ml boxes (Greiner Bio-One). Absorbance at 280 nm was recorded for each fraction. Relevant fractions containing the purified hybrid were pooled, quantified and concentrated by ethanol precipitation and analysed by native PAGE.

From May 2015 to April 2016, we carried out a cross-sectional study among hospitalized children under 5 years of age, presenting with acute gastroenteritis in the following hospitals in the Littoral region of Cameroon; the Douala Laquintinie hospital, the district hospitals of Bonassama and Deido, and the regional hospitals of Edéa and Nkongsamba. A diarrheic stool sample was collected from each participating hospitalized child along with socio-demographic information including RVA immunization and breastfeeding status which were obtained from the patient medical record and/or from guardians. The stool specimens were transported in a cooler (4 °C to 8 °C) following recommended standard to the virology laboratory of the Mother and Child Centre of the Chantal Biya’s Foundation, Yaoundé, and stored in aliquots of 1.5 mL at − 80 °C until analyzed. Prior to storage, a 10% (v/v) suspension to be used for total RNA extraction was prepared for each sample using nuclease-free distilled water (Invitrogen™) as diluent, then stored at − 20 °C until used.