Ms2 bacteriophage rna

QIAamp viral RNA mini kit (Qiagen, Valencia, CA) was used in the purification of nucleic acids without DNase I treatment step from clear supernatants of viral cultures. Carrier RNA (Qiagen) was used to protect RNA sample from degradation. Bacteriophage MS-2 RNA (Roche Diagnostics Corporation, Indianapolis, IN) was added as an internal control.

The miRNeasy serum/plasma kit (Qiagen, Venlo, Nederland) was used according to the manufacturer’s instructions to extract miRNAs from human serum samples. For each 200-μL serum sample, 1.6 x 10⁸ copies of C. elegans (Ce)-miR-39-1 (Qiagen) was used as non-mammal spike-in control and 1 μg bacteriophage MS2 RNA (Roche) was used as carrier RNA. The miScript II reverse transcription kit, the miScript SYBR^® Green PCR kit (Qiagen), and a Roche Lightcycler (Roche) were used for qRT-PCR amplification of serum miRNA.

miRNA extraction was performed for each serum sample using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. As a first step, synthetic RNA Cel-miR-39 (Qiagen) from C. elegans was added in the quantity recommended by the manufacturer as a quality control to determine the efficiency of the extraction and for miRNA normalization because of the absence of homologous sequences in humans, with 1 μg of bacteriophage MS2 RNA (Roche, Basel, Switzerland) also added to each sample to act as a carrier.
Briefly, serum samples were thawed and centrifuged for 5 min at 3000×g; 200 μl of the supernatant were used for miRNA extraction and afterwards Cel-miR-39 and MS2 RNAs were added. Subsequently, extraction was performed following the manufacturer’s instructions. Total miRNA was eluted in 40 μl H₂O and stored at – 80 °C until use.

Total RNA was extracted from 200 µl of serum using spin column chromatography (miRNeasy Mini kit, Qiagen GmbH, Hilden, Germany) as previously reported^{35 (link)}. One μg of MS2 bacteriophage RNA (Roche Life Science, Mannheim, Germany) and 5 μl of 5 nM synthetic spike-in, Caenorhabditis elegans-miR-39 (C.el. miR-39-3p) were added to the samples during processing, according to the manufacturer’s protocol. The inclusion of the synthetic miRNA was necessary for normalization of sample-to-sample variation in RNA isolation. RNA was eluted by adding 15 µL of RNase-free water, and 4 µl was subjected to retro-transcription using Universal cDNA synthesis kit II (Exiqon Inc., Vedbaek, Denmark) in a total of 20 µl, including 1 µl of synthetic spike-in UniSp6. To avoid variation due to sample differences and handling, all variables were kept consistent throughout the study. The amount of RNA in serum is very low, and the concentration could not be accurately determined in these samples. Consequently, for the qPCR analysis, a constant input amount was used for all samples, using the same starting volume rather than RNA quantity. The uniformity of the RNA extraction and efficiency of the RT and PCR reactions were analyzed monitoring the synthetic spike-in RNA templates.

Total RNA was extracted from 200 μl of EV samples using a miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) for RT-qPCR, according to the manufacturers’ instructions. To improve the RNA yield, 1 μg of RNA carrier (MS2 bacteriophage RNA [Roche Applied Science]) was applied, and 2 fmol of a synthetic C. elegans miRNA cel-miR-39 (1 μl of 2 nM) (Qiagen) was added to denatured samples for normalization of sample-to-sample variation. Total RNA was eventually eluted by adding 60 μl of RNase-free water. Next, total RNA samples were reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and each miRNAs primer. Other primers for gene expression assay can be also found in the Supplementary Methods. RT-qPCR was performed using TaqMan Fast Advanced Master Mix and a LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) in 96-well plates. Amplification curves were analyzed using the Roche LC software program, and were used to establish PCR amplification efficacy. All reactions were performed in duplicate.

Immediately after the collection of tissue biopsies, the specimens were placed in RNAlater™ (Sigma Aldrich, St. Louis, MO) and stored at -80°C for further RNA isolation.
Total RNA including miRNAs were isolated using miRCURY RNA Isolation Kit Tissues (Exiqon, Vedbaek, Denmark) according to the manufacturer's instructions. The quantity and purity of RNA were analyzed spectrophotometrically, and 100 ng of RNA was used for cDNA synthesis, which was synthesized using the Universal cDNA Synthesis Kit II (Exiqon). Spike-in UniSp6 was added to each sample, which allowed to monitor the quality of reaction with reverse transcriptase. Three negative controls of this reaction were prepared using the following reaction mixtures—without reverse transcriptase (no enzyme sample), without RNA template (no template), and using MS2 bacteriophage RNA (Roche, Cat. No. 10165948001) as the template (Mock).
One microgram of total RNA isolated from tissue samples, as mentioned above, was reverse transcribed using the Transcriptor Universal cDNA Master (Roche Diagnostics, Mannheim, Germany) and subsequently used for the evaluation of the expression of CDH1. Negative control samples were prepared with the omission of an enzyme and RNA template.

Optimization of the RNA extraction procedure was tested on four seminal plasma samples. For isolation of total RNA from seminal plasma samples, two procedures for extraction were compared: the miRCURY RNA isolation kit—Biofluids (#300112; Exiqon, Vedbaek, Denmark) and a combination of TRIzol (Invitrogen, Carlsbad, CA, USA) followed by Nucleospin columns (#740955; Macherey-Nagel, Düren, Germany). Protocols, including the incorporation of proteinase K and MS2 bacteriophage RNA (#10165948001; Roche) to the Biofluid kit method, were carried out according to the supplier’s manuals and a previous paper [16 (link)]. Proteinase K was added to the seminal plasma in a final concentration of 2 µg/µL and incubated for 10 min at 37 °C. The input volume of seminal plasma in the RNA extraction kits was tested between 25, 50, 100, and 200 µL. To test whether exosome isolation prior to RNA extraction leads to higher microRNA yields, 50–100 µL of seminal plasma was processed by the miRCURY exosome isolation kit—serum/plasma (#300101, Exiqon, Vedbaek, Denmark). The RNA obtained from each extraction procedure was dissolved in 50 µL of RNAse-free water.