Tripure reagent

RNA was isolated with TriPURE reagent (Roche) according to the manufacturer's protocol. Reverse transcription with random hexamer primers was done with Transcriptor cDNA First Strand Synthesis Kit (Roche). PGR gene and PR-dependent genes expression analyses were carried out with TaqMan (Applied Biosystem) probes -PGR (Hs01556702_m1), CHN2 (Hs00906968_m1), RGS2 (Hs01009070_g1) with ACTB (Hs99999903_m1) and GAPDH (Hs99999005_m1) used as reference genes. Twenty microliter reactions were conducted applying TaqMan Universal PCR Master Mix (Applied Biosystem) on 96-well plates in CFX96 cycler (Bio-Rad, Hercules). Reactions were done in duplicates. Each plate contained an inter-run calibrator - a set of non-template controls and controls for cDNA contamination. Gene expression was calculated using a modified ΔΔC approach, as previously described [59 (link)].

For quantification of endogenous or transfected miR-15a and miR-16 expression levels total RNA was isolated using TRI Pure reagent (Roche, Indianapolis, IN 46250-0414 USA), quantified, 1 μg RNA was reverse transcribed and quantitative polymerase chain reaction was performed using TaqMan® miRNA assays (Cat No. 000389 for hsa-miR-15a, Cat No. 000391 for hsa-miR-16, and Cat. No 001973 for U6; from Life Technologies, Grand Island, NY, US). Relative miR expression levels were calculated using the comparative C_t method with U6 as the normalizer [6 (link)].

Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostics, Barcelona, Spain) and then purified with E.Z.N.A. Total RNA Kit I (Omega Biotek, Vermont, USA), following the manufacturer’s instructions. Isolated RNA was quantified using a NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was confirmed using agarose gel electrophoresis.

Blood from 4 h fasted volunteers was collected using Vacutainer EDTA tubes. After blood collection, PBMC were isolated using Ficoll-Paque Plus density gradient media (GE Healthcare Bio Science, Madrid, Spain). In brief, the anticoagulant treated blood was diluted with an equal volume of a phosphate-buffered saline (PBS) solution. Next, the blood was layered carefully over Ficoll without intermixing (10 mL of Ficoll for 20 mL of blood mixed with PBS) in a centrifuge tub and centrifuged at 400g for 30 min at 20 °C in a swinging bucket rotor with acceleration and deceleration adjusted at zero. PBMC, together with platelets, were harvested from the interface between Ficoll and plasma layers. This layer was then centrifuged in PBS at 300g for 10 min at 20 °C to wash PBMC and to remove the platelets. PBMC pellet was finally suspended in Tripure Reagent (Roche Diagnostics, Barcelona, Spain) for its storage at – 80 °C. According to our previous evidences, approximately 84% of the cells obtained with this isolation method are lymphocytes and 16% monocytes, with no differences between NW or OW-OB subjects^{24 (link)}.

Total cellular RNA was isolated from 15-day-old F. velutipes mycelia using Tripure reagent (Roche) according to manufacturer’s instructions. Total RNA was separated on 1.5% agarose-formaldehyde gel and transferred onto a nylon membrane by capillary blotting. Equal loading was confirmed by Ethidium Bromide and Methylene blue staining. Levels of mRNA on autoradiograms were quantified by densitometry scanning using Flour-S-(Bio-Rad) software.
For Southern blot analysis, genomic DNA was isolated by cetyltrimethylammonium bromide (CTAB) method. 10μg of digested DNA was separated on 0.8% agarose gel and Southern blot analysis was performed as described earlier [44 (link)]. High stringency post-hybridization washes were performed at 65°C with 2 X SSC, 1% w/ v SDS and with 0.2 X SSC, 1% w/ v SDS. Blots were probed using [γ-P³²] ATP labeled FvC5SD cDNA or gene as probe.

Total RNA was isolated using the TriPure reagent (Roche) and retro‐transcribed (50 ng) using Transcriptor Reverse Transcriptase (Roche) and random hexamers according to the manufacturer's instructions. Quantitative PCR was performed using the Precision 2X QPCR master mix (Primer Design) and the Realplex II Mastercycler (Eppendorf) according to the manufacturer's instructions. Both LINC00152‐specific primers (forward: 5′‐TTC ACA GCA CAG TTC CTG GG‐3′ and reverse 5′‐GGG GGC TGA GTC GTG ATT TT‐3′) and housekeeping U6 primers (forward: 5′‐CTC GCT TCG GCA GCA CA and reverse: 5′‐AAC GCT TCA CGA ATT TGC GT‐3′) utilized for PCR reactions have been synthesized by TIB Molbiol (Genoa, Italy). OSU‐CLL cell line was obtained from the Ohio State University and previously described.²⁷ MEC1 cell line was previously described.²⁸ siRNA designed to knock down LINC00152 was 5′‐CUAUGUGUCUUAAUCCCUUtt‐3′ (Ambion). Control silencing was performed using the commercial control siRNA‐A (Santa Cruz, # sc‐37007). Transfections were carried out for 48 h using Kit V for Nucleofector II (Amaxa) according to the manufacturer's instructions.