Stool total rna purification kit

We selectively evaluated the presence of viruses with human pathogenic potential—i.e., Adenovirus (ADV), Cytomegalovirus (CMV), Human Herpesvirus (HHV) 6A and 6B, and 8 and Enterovirus (ETV)—in cryopreserved stool DNA and RNA aliquots using commercial RT-PCR kits and following the manufacturer’s instructions, including adjustment of quantification using a sensitivity control at 1 copy/μl. For ADV, the Adenovirus R-gene® kit (BioMérieux, Marcy-l’Étoile, France) was used. For CMV, HHV 6A, 6B, and 8, specific probe/primer mixes for RT-PCR assays (Virusys Corporation, TaneyTown, MD, USA) were used in combination with the TaqMan® Gene Expression Master Mix (Thermo Scientific, DE, USA) at 1/20 dilution. For ETV, fecal RNA was first extracted from RNAlater®-cryopreserved fecal samples using the Stool total RNA Purification Kit (Norgen Biotek, Corporation, Thorold, Canada). Non-diluted fresh RNA aliquots were directly used to perform qualitative RT-PCR testing of enteroviruses using the Enterovirus R-gene® Kit (BioMérieux, Marcy-l’Étoile, France).

Before surgery or any treatment, 5 mL of blood were collected from each patient and placed in an S-Monovette^® 9 mL, Serum Gel with Clotting Activator tube (Sarstedt, Nümbrecht, Germany). RNA was extracted from the serum samples using the miRNeasy^® Serum/Plasma Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, as previously described [19 (link)]. RNA was extracted from the stool samples with a Stool Total RNA Purification Kit (Norgen Biotek Corp.), as described by Tarallo et al. [86 (link)].

All vaginal samples underwent RNA isolation using the Stool total RNA purification kit (Norgen, Thorold, ON, Canada). The total RNA was treated with RNase-free DNase I (Roche, Almere, Netherlands; 10 U of DNase/20 μg of RNA) for 20 min at room temperature. The quality and concentration of the RNA extracts were determined using 1% agarose–0.5× TBE gels and spectrophotometric measurements at 260, 280, and 230 nm obtained using a NanoDrop® ND-1000 spectrophotometer. The total RNA extracted was retrotranscribed to complementary DNA (cDNA) using random hexamers and a Tetro cDNA synthesis kit (Bioline, Freiburg, Germany), according to the manufacturer’s instructions. The obtained cDNA was subjected to Ion Torrent 16S rRNA gene-based analysis.

Total RNA was extracted from 200 µl faecal aliquots with the Stool total RNA purification kit (Norgen Biotek Corp) using the protocol recommended by the manufacturer. RNA quality and quantity were verified according to the MIQE guidelines (http://miqe.gene-quantification.info/). For all samples, RNA concentration was quantified by Qubit fluorometer with a Qubit microRNA assay kit (Invitrogen).

The 3 aliquots of feces collected during the experiment from each volunteer were pooled together for nucleic acid extraction. Ten g of the pool was aseptically homogenized with 90 ml of Ringer’s solution (Oxoid) for 2 min in a stomacher (PBI) at room temperature for DNA extraction. An aliquot of two ml was collected and centrifuged at the maximum speed for 30 s, then the supernatant was removed and the DNA was extracted from the pellet using a Powersoil DNA kit (MO-BIO, Carlsbad, CA, USA), according to the manufacturer’s instructions. DNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Milano, Italy) and standardized at 50 ngμl^-1. About 250 mg of the feces pool was directly subjected to RNA extraction using a Stool Total RNA Purification Kit (Norgen Biotek Corp. Ontario, Canada), according to the supplier's instructions. Seven μl of TURBO-DNase (Life Technologies) was added to digest the DNA in the RNA samples, with an incubation of 3 h at 37°C. RNA was quantified, as described above, and standardized at 500 ngμl^-1. Each RNA solution was checked for the presence of residual DNA by performing PCR amplification. When positive signals were detected, the DNase treatment was repeated.

Total genomic bacterial RNA was isolated from frozen stool samples using the Stool Total RNA purification KIT (Norgen Biotek Corp., Ontario, Canada), according to the manufacturer’s instructions. The quality and concentration of the RNA were determined by spectrophotometric measurements at 260, 280, and 230 nm through the NanoDrop ND-1000 spectrophotometer. Total RNA extracted (approximately 2.5 μg) was retrotranscribed in cDNA using random hexamers and the Tetro cDNA synthesis kit (Bioline USA, Inc., Taunton, MA, USA), according to the manufacturer’s instructions.
Bacterial Microbiome was estimated by 16S rRNA. A 16S metagenetic analysis was carried out at Genomix4life (spin-off of the University of Salerno, Fisciano, Italy) using the Illumina MiSeq platform. The V3–V4 region of the 16S rRNA gene was amplified for analysis of diversity inside the domains of bacteria [31 (link)]. PCR and sequencing analyses, quality control, and taxonomic assignment were carried out according to the protocol of Genomix4life. Shannon diversity and alpha diversity indices were calculated using Qiime analysis [32 (link)].