Qiacube extractor

Extraction of total RNA from stool, urine, plasma EVs, and tissues was performed using appropriate kits/methodologies for total RNA purification according to the specimen to be analyzed.
RNA from tissues was isolated using QIAzol (QIAGEN) after tissue homogenization performed with ULTRA‐TURRAX® Homogenizer,¹³ followed by phenol/chloroform extraction according to the manufacturer's standard protocol.
Total RNA from stool samples was extracted with the Stool Total RNA Purification Kit (Norgen Biotek Corp.) according to the manufacturer's protocol. Total RNA from plasma EVs was extracted with the miRNeasy Plasma/Serum Mini‐kit (QIAGEN) using the QIAcube extractor (QIAGEN). Total RNA from urine samples was extracted with the Urine microRNA Purification Kit (Norgen Biotek Corp.), following the manufacturer's standard protocol. The RNA concentration was quantified by Qubit™ 4 fluorometer with Qubit™ microRNA or RNA Broad range Assay kits (Invitrogen).

Blood samples were collected according to standard phlebotomy procedures at the moment of the recruitment. Samples were collected into ethylenediaminetetraacetic acid (EDTA) tubes and immediately processed for plasma separation with centrifugation at × 1000 g for 10 min at room temperature. Once separated from the rest of blood, plasma was distributed in cryovial tubes. One tube was immediately used for RNA extraction while the other aliquots were labelled and stored at −80°C. The time interval between sample collection, processing and storage at −80°C was less than 3 h.
Total RNA was extracted from 200 µL of plasma with the miRNeasy plasma/serum mini kit (Qiagen) using the QiaCube extractor (Qiagen) following the manufacturer's instructions. RNA samples were stored until analysis at −80°C.

EVs were isolated from 200 µL of serum using ExoQuick precipitation solution (System Biosciences, Palo Alto, CA, USA) according to manufacturer’s instructions. Briefly, 200 µL of serum was processed with 50.4 µL ExoQuick solution and stored at 4 °C overnight. The EVs pellets were resuspended with 200 µL of nuclease free water and RNA was immediately isolated from the solution.
Total RNA was extracted with miRNeasy serum/plasma kit (Qiagen, Hilden, Germany) using the QIAcube extractor (Qiagen, Germany) according to manufacturer’s instructions. RNA concentration was determined for all samples with Qubit 2.0 Fluorometer with miRNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

A total of 38 DNA samples consisting of controls, probands and their parents were recruited by the University Hospital “G. Rodolico—San Marco” in Catania, Italy. The test group consisted of 18 patients (10 females and 8 males), with suspected HPA based on abnormal blood levels of Phe, and 20 controls (18 were parents). The biochemical and clinical parameters of the patients are reported in the Supplementary Materials (S1). This study’s protocol (1327/2020) was approved by the Institutional Review Board, and written informed consent was obtained from all participants or their legal tutors. All methods were performed in accordance with ethical regulations.
Genomic DNA was isolated from dried blood (Guthrie) spots using a QIAamp UCP DNA micro-Kit on an automatic Qiacube extractor (Qiagen, Germantown, MD, USA). The quantification of DNA was assessed by measuring the genomic copies of the human RNase P gene using the TaqMan^® RNase P Detection Reagents Kit (ThermoFisher Scientific, Waltham, MA, USA) on the Aria Dx Real-Time PCR system (Agilent Technologies, Santa Clara, CA, USA).

Total RNA from plasma exosomes was extracted with the miRNeasy plasma/serum mini kit (Qiagen) using the QiaCube extractor (Qiagen). RNA from stool was extracted using the Stool Total RNA Purification Kit (Norgen Biotek Corp). Total RNA from urine was extracted with Urine microRNA Purification kit (Norgen biotek corp), following the manufacturer’s standard protocol.
RNA from cervical scrape was extracted from samples stored in STM or RNA-later, using the miRCURY™ RNA Isolation Kit - Cell & Plant (Exiqon) following manufacturer`s protocol.
RNA quality and quantity was verified according to MIQE guidelines (http://miqe.gene-quantification.info/). For all samples, RNA concentration was quantified by Qubit^® 2.0 Fluorometer with Qubit^® microRNA Assay Kit (Invitrogen).