Separation of the paramagnetic beads was performed with a neodymium magnet (SuperMagnete, Gottmadingen, Germany). Prior to use, 20 µL DynaBeads oligo-dT25 magnetic bead solution (Life Technologies) was aliquoted into a 200 µL PCR tube (Thermo Fisher Scientific, Hvidovre, Denmark) and washed once in the PDE, Caspase-3 or LDH lysis buffer (native protein protocol; NP) for the respective enzyme assay or in the LiDS lysis buffer for the standard denaturing oligo-dT bead protocol (SDP). Each ovary was homogenized in 200 µL lysis buffer by sonication 2 × 30 s in a Branson Sonifier 250 with output set at 4% and 50% duty cycle in an ethanol ice bath. For the caspase-3 assay, each sample containing hGCs (104-105 cells) was suspended in 9 µL H2O and divided into 3 tubes with 40 µL lysis buffer (buffer APL from the AllPrep RNA/Protein kit; Qiagen, Copenhagen, Denmark). For determining the optimal NaCl concentration for mRNA isolation, 105 hFFs were lysed in 200 µL Caspase-3 lysis buffer containing 0, 150, 300, 500, or 1000 mM NaCl. The lysate was spun down for 30 s at 13,000 × g, and the supernatant was transferred to the washed magnetic beads. The mRNA was annealed to the beads for 20 min on a rotating mixer at 4°C (NP) or for 5 min at room temperature (SDP), followed by magnetic separation of the protein-containing liquid phase and mRNA-containing beads. The liquid was snap-frozen and stored at -80°C for further enzymatic assays, and the beads were washed twice in 200 µL wash buffer A, twice in 200 µL wash buffer B, once in 200 µL 1× RT buffer, and finally, the mRNA was eluted from the beads in 40 µL Tris-HCl for 2 min at 75°C.
For comparison, total RNA was isolated according to the manufacturer's instructions using the RNeasy Mini Kit (Qiagen), AllPrep RNA/Protein kit, GenElute Mammalian Total RNA Miniprep Kit (Sigma- Aldrich), or TRIzol Reagent (Life Technologies), followed by further RNA purification with the RNeasy kit.First-strand synthesis
First-strand cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Briefly, a master mix containing 3.0 µl 10× RT Buffer, 1.2 µl 25× dNTP mix (100 mm), 3.0 µl 10× RT Random Primers, 1.5 µl MultiScribe Reverse Transcriptase (5.0 U/µl), and 1.3 µl nuclease-free (DEPC-treated) water was prepared, and for each 30 µl reaction, 10 µl master mix was added to 20 µl purified RNA. Negative controls were prepared by substituting MultiScribe Reverse Transcriptase with nuclease-free water. First strand cDNA for 3′:5′-end quality control was synthesized by replacing RT random primers with anchored oligo-dT primers (Thermo Fisher Scientific). All steps were performed on ice. Samples were centrifuged briefly at 12,000 × g and then incubated at room temperature for 10 min, followed by 37°C for 2 h, and finally, 85°C for 5 s. First-strand cDNA was stored at -20°C until qPCR analysis.Real-Time PCR
The RNA yields were evaluated by real-time quantitative PCR using TaqMan technology (Life Technologies). Assuming equal PCR efficiency, variations in the Cq values would arise from differences in the quality or starting amount of RNA since the RNA was eluted in the same volume for all of the kits tested. The Cq values were estimated using the second derivative maximum method. All TaqMan Gene Expression Assays (pre-designed) were purchased from Life Technologies (Table 1). Sample duplicates (negative controls) were prepared according to the manufacturer's instructions. A total reaction volume of 10 µL was used. cDNA was amplified using the LightCycler 480 qPCR instrument (Roche, Hvidovre, Denmark) using the following thermal cycling conditions: 95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min.Table 1.
Immunoprecipitation and Western blot
Immunoprecipitation was carried out on 100 µL of hCL homogenate containing 320 µg total protein in immunoprecipitation lysis buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1% Protease Inhibitor Cocktail, and 137 mM NaCl. Lysis buffer for RNA extraction also contained 500 mM NaCl and 5 mM DTT. Incubation with oligo-dT beads was done as previously described, and the beads were discarded afterwards.
The lysate was pre-cleared with 50 µL Exosome Immunoprecipitation Dynabeads, protein-G (Life Technologies)for 10 min followed by a 1 h incubation with primary rabbit antibodies against GAPDH, β-actin (AbCam, Cambridge, UK), or solid-phase absorbed rabbit immunoglobulin fraction (DAKO, Glostrup, Denmark) and a 20 min incubation with 50 µL Exosome Immunoprecipitation Dynabeads, protein-G. The supernatant was removed, and the beads were washed 3 times in PBS with 0.05% Tween-20 (PBST, Merck) before the antibody and antigen were eluted by 10 min at 70°C in 20 µL NuPage LDS Sample-buffer (Life Technologies) with 100 mM DTT. The immunoprecipitate was separated by electrophoresis on a NuPAGE 12% Bis-Tris polyacrylamide gel (Life Technologies) in MOPS SDS running buffer (Life Technologies) and transferred by electroblotting to an Invitrolon PVDF membrane (Life Technologies). The membrane was incubated 30 min in Western blocking buffer and subsequently in primary antibody (anti-GAPDH 0.2 µg/ mL or anti-β-actin 0.2 µg/mL) diluted in blocking-buffer overnight at 4°C. After washing three time in PBST, the membrane was incubated for 1 h in donkey anti-rabbit IgG HRP-conjugated secondary antibody (0.2 µg/mL) (Agrisera, Vännas, Sweden) diluted in blocking-buffer. The membrane was washed three times in PBST and detected by chemiluminescence using an ECL reagent (8) in a ChemiGenius2 station (Syngene, Frederick, MD).