Ms2 carrier rna

Total RNA was isolated from islets using the Qiagen RNeasy Mini Kit as per the manufacturer’s instructions, and reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Quantitative RT-PCR was performed using Ssofast EvaGreen Supermix (Bio-Rad) with Hprt1 as the reference gene, or TaqMan Fast Universal PCR Master Mix for Ins1 and Ins2 (Applied Biosystems) with mouse β-actin (4352341E, Thermo Fisher Scientific) as a reference gene, in a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). Transcript levels were quantified by the 2^−ΔΔCT method. All primer sequences are listed in Supplementary Table S2.
For miR-375 measurement in the circulation, extracellular RNA was isolated from plasma samples in the presence of an MS2 carrier RNA (Roche, Laval, Canada) using the Qiagen miRNeasy Kit. Reverse transcription was performed with the Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark). Quantitative RT-PCR was performed using the ExILENT SYBR Green Master Mix (Exiqon) with LNA-based miRNA primers (Exiqon). Relative values were calculated with the ΔCT method using miR-16* as a reference miRNA.

For all analyses, RNA was extracted from 200 μL of serum using the miRNeasy serum kit (Qiagen) into a final volume of 100 μL nuclease free water as described previously (12 (link)). All isolated RNA was stored at -80°C. RNA yield was maximised with the addition of MS2 carrier RNA (Roche, final concentration 1.25 μg ml⁻¹) to Qiazol prior to isolation and the exogenous spike-in miRNA cel-miR-39-3p (5.6 × 10⁸ copies) was used as an initial quality control for extraction efficiency. All samples underwent quality control analysis prior to subsequent target miRNA qRT-PCR analyses (12 (link), 16 (link)). Quality control analysis included quantification of cel-miR-39-3p, the endogenous housekeeper miR-30b-5p and the haemolysis control miRNAs miR-451a and miR-23a-3p as previously described (12 (link)). Consistency of extraction was acceptable for all samples analysed (14 (link)). Haemolysis assessment was performed by calculating the delta Ct values for miR-23a-3p minus miR-451a, detailed in (14 (link)).

Briefly, we followed RNase-free protocols throughout all procedures up to qPCR setup (after RNA has been converted to cDNA). Blood samples were collected in 4.0 ml serum tubes (VWR Cat. No. CABD 367812) and were spun down in a refrigerated centrifuge within 2 hours of collection. Using a transfer pipette, the serum was removed into a cryogenic vial and gently mixed by drawing the serum up and down in the tube several times. Once mixed, the serum was aliquoted into six 1.2 ml-cryogenic vials. The aliquots of serum were stored in -80 C freezer until selected for analysis. RNA purification was conducted on 200 μl of serum samples. Total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Due to the low serum RNA yield, 1 μg MS2 carrier RNA (Roche, Switzerland) was added during RNA purification steps in order to maximize the RNA yield and minimize purification efficiency variation.

miRNAs were isolated from 50 µL serum and plasma using target-specific anti-miR magnetic beads, as reported before (OncoTarget 2016). In short, a KingFisher Flex robot with TaqMan® miRNA ABC Purification Kit Human Panel A (ThermoFisher PN 4473087, Waltham, MA, USA) was used to isolate miRNAs. All reagents are provided in the kit. These panels consist of superparamagnetic Dynabeads covalently bound to a unique set of ~380 anti-miR oligonucleotides. Briefly, 100 µL of lysis buffer (containing spike-in) was added to 50 µL of serum/plasma, followed by the addition of 80 µL of beads (10⁶ beads/µL). Samples were incubated at 30 °C for 40 min, then washed three times with wash buffer. The bound miRNAs were eluted from the beads with 100 µL elution buffer.
RNA from 200 µL of thawed serum and plasma was isolated using the miRNeasy Serum/Plasma Advanced Kit from Qiagen (PN 217204), according to the manufacturer’s instructions. In order to increase RNA yield, MS2 carrier RNA (Roche PN 10165948001) was added to a final concentration of 1.25 µg/mL. Total RNA was eluted from columns with 50 µL of nuclease-free water. During the lyses of the samples, a non-human spike-in Cel-miR39 (5.6 × 10⁸ copies) external control was added to each sample, in both isolation-techniques, to monitor the RNA recovery.

Total RNA from serum/plasma was isolated using miRNeasy serum/plasma isolation kit (Qiagen) according to manufacturer’s protocol. Briefly, 20 µl serum/plasma was added with 180 µl phosphate-buffered saline (PBS). 1 ml QIAzol lysis reagent was spiked with a) 1 µg MS2 carrier RNA (Roche) to reduce loss of RNA during isolation and b) a set of 3 synthetic small RNAs (MiRXES, Singapore) with sequences distinct from any of the 2588 annotated mature human miRNAs (miRBase version 21) to monitor RNA isolation efficiency. This QIAzol mixture was aliquoted to the 200 µl sample for lysis. 200 µl chloroform was then added, thoroughly mixed and centrifuged at 18,000 g for 15 minutes (min). 600 µl aqueous phase was subsequently removed for spin-column based purification and RNA was eluted in 30 µl nuclease-free water.