Mirneasy serum plasma mini kit

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.

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).

Serum miRNA was purified using a miRNeasy Serum/Plasma Mini Kit (#217004, Qiagen, Venlo, the Netherlands) according to the original manufacturer’s protocol. The RT reaction of miRNA for each sample was performed using a miScript II RT kit (#218161, Qiagen) to obtain complementary (c)DNA based on the manufacturer’s instructions.

Total RNA was isolated from serum using the miRNeasy Serum/Plasma mini kit (Qiagen, Hilden, Germany). Briefly, 800 μL serum was lysed with 5 volumes of Qiazol and processed according to the manufacturer’s instructions. Glycogen (Thermo Fisher Scientific, Waltham, MA, USA), 10 μg, was added to each sample lysate to increase RNA yield. RNA quantification was performed by Nanodrop 1000 (Thermo Fisher Scientific). Total RNA was finally eluted in 30 μL RNase-free water and stored at −80 °C until analysis.

Plasma, isolated from the whole blood as previously described (Section 2.4), was processed by using the miRNeasy Serum/Plasma Mini Kit (Qiagen, Hilden, Germany) to isolate total RNA, including microRNAs (miRNAs). Retrotranscription was carried out using the miRCURY LNA miRNA RT Kit (Qiagen, Hilden, Germany), and the obtained cDNA was diluted to 1 : 30, immediately before use. Real-time PCR was run on the MiniOpticon CFX 48 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the miRCURY LNA miRNA SYBR Green PCR and specific miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany), as previously reported [57 (link)]. The miRCURY Primer Assay specific for hsa-miR-195-5p (MIMAT0000461), hsa-miR-153-3p (MIMAT0000439), and hsa-miR-93-5p (MIMAT0000093) was purchased from Qiagen (Hilden, Germany).
The relative miRNA expression was calculated using the Ct method and normalized on miR-93-5p. Several pieces of evidence reported high stability of miR-93-5p in biofluids [58 (link)–61 (link)]; thus, miR-93-5p was suggested as a plasmatic reference gene in the manufacturer's handbook. According to this, the expression levels of miR-93-5p in plasma samples of our cohort showed comparable expression levels without significant difference among groups (data not shown) [49 (link)].

RNA was isolated using the miRNeasy Serum/Plasma Mini Kit (Qiagen, Hilden, Germany). The manufacturer's protocols were followed as written, except for the final collection step which was performed sequentially in two steps. First, 25 μL of nuclease-free water was added to the spin column for 10 min before elution of sample. Then, this step was repeated using 15 μL of nuclease-free water to increase the recovery yield. Combination of both eluates yielded around 30 μL of total RNA collected per pooled exosome sample from five mice. The amount and quality of RNA was analyzed using a Nanodrop to assess initial concentration, and TapeStation (Agilent) to assess sample quality utilizing RNA integrity number (RIN).