Zeta probe nylon membrane

For isolation of poly(A) RNA from total RNA, Oligo d(T)25 magnetic beads (New England Biosciences) were used in combination with a 12-tube magnetic rack. Beads were washed once in a binding buffer/wash buffer (20 mM Tris-HCl at pH 7.5, 500 mM LiCl, 1 mM EDTA) similar to manufacturer recommendations except that DTT was left out of the buffer, as this would cleave the RNA conjugated to biotin-HPDP. At least 40 µg of total RNA was added to 200 µL of beads. Samples were washed in 1x binding buffer, then 1x low-salt buffer, and eluted from beads in TE buffer following incubation for 3 min at 50°C.
For each sample, 200 ng of mRNA was blotted onto a Zeta-Probe nylon membrane (BioRad) using a BioRad DotBlot. The RNA was cross-linked using a UV cross-linker. The blot was blocked using blocking buffer (PBS, 10% SDS, 1 mM EDTA) for 20 min. Samples were then probed with Streptavidin Alkaline phosphatase in blocking solution (1:1000). The membrane was washed in PBS at decreasing concentrations of SDS (10%, 1%, 0.1%) for 10 min each. Spots were visualized using ECF substrate (GE Healthcare), visualized on a Typhoon FLA 9500, and analyzed using ImageQuant software.

Total RNA from cj319-WT marmoset cells or 293T/17 cells transfected with wild type or mutant versions of pBS-GFP-HSUR2 plasmids and stored in 1 ml of TRIzol was prepared following the manufacturer's protocol (Ambion). RNA samples were then suspended in 20 µl (marmoset cells) or 50 µl (293T/17 cells) of 2 mM Tris-HCl, pH 7.5, 8 M urea and 20 mM EDTA and heated at 65°C before loading onto a 6% denaturing polyacrylamide gel (only 25 µl of each 293T/17 cell sample were loaded onto the gel). Gels were run at 10 W in 1X TBE and transferred onto a Zeta-Probe nylon membrane (Bio-Rad) for 30 min at 1 A using a semi-dry blotting unit (Fisher Biotech). The membranes were pre-hybridized at 42°C in ExpressHyb hybridization solution (Clontech) for 30 min. Hybridization of ³²P-labeled probes to HSUR2, HSUR7 and U6 was performed in ExpressHyb solution overnight at 42°C. Blots were washed once with 2X saline sodium citrate (SSC) buffer, 0.1% SDS for 15 min at room temperature followed by a wash with 0.5X SSC, 0.1% SDS for 15 min at room temperature. Membranes were wrapped in saran wrap and exposed to a phosphorImager screen (GE Healthcare).

Separation of total RNA (10 μg) was performed on 8% or 12% polyacrylamide gels in TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA). As a denaturing agent, 1% bleach was used to replace 7 M urea. The separated RNAs were then transferred to a positively charged Zeta-Probe nylon membrane (Bio-Rad), using semi-dry electroblotting (Bio-Rad). RNAs were covalently cross-linked to the nylon membranes at 1200 mJ in a UVC-508 Ultraviolet Crosslinker (Ultra-Lum Inc., Carson, CA). ssDNA oligonucleotides (See S1 Table) were labeled with ³²P-γATP using Polynucleotide kinase (Fermentas/Thermo Scientific), according to instructions of the manufacturer. The membranes were incubated overnight for hybridization at 42°C in PerfectHyb Plus Hybridization buffer (Sigma-Aldrich Chemie Gmbh, Munich, Germany) with 9 μl 0.16 pmol/μl of the labeled probe. Membranes were washed twice in 2x saline sodium citrate (SSC) buffer with 0.1% SDS, after which they were exposed to a Phosphor Screen overnight. A Cyclone Plus Phosphor Imager and OptiQuant software (PerkinElmer, Groningen, NL) was used for imaging.

Total plant DNA was extracted [27 ] and estimated by fluorometry. DNA was digested with suitable restriction enzymes and electrophoresed in a 0.8% (w/v) agarose gel in 1 X TNE (40 mM Tris-acetate, pH 7.5, 20 mM sodium acetate, 2 mM EDTA) or 1 X TBE (Tris-borate-EDTA) buffers. DNA was transferred [28 (link)] to the Zeta-probe nylon membrane (Bio-Rad, Hercules, CA, USA). DNA was labeled with [α-³²P]dCTP using the Megaprime DNA labeling system (GE Healthcare UK Ltd., Little Chalfont, UK) to prepare probes. Hybridizations were carried out at 65°C and high stringency post hybridization washes were performed [29 (link)].

Northern blot analysis was carried out as previously described (Andreassen et al., 2018 (link)). Briefly, 10 μg of RNA was run on a denaturing 8% polyacrylamide gel for 2 h at 300 V. Separated RNA was blotted onto a Zeta-probe nylon membrane (Bio Rad) using a semi-dry transfer unit for 1 h at 400 mA. RNA was cross-linked to the membrane with UV radiation. Oligonucleotide probes (see Supplementary material; Supplementary Table S1) were 5′-labeled with³² P-ATP using T4-polynucleotide kinase (New England Biolabs). Membranes were pre-hybridized for 10 min at 42°C before probing with 5′-labeled oligos overnight. Probed membranes were washed once in 2× SCC and 0.1% SDS for 10 min followed by a 10 min wash in 0.5× SCC and 0.1% SDS. Probed membranes were visualized by phosphorimaging on a Typhoon scanner (GE Healthcare).

Total RNA was prepared from dissected tissues of C57BL/6 mice using Tri reagent (Molecular Research Center, Inc.). Total RNA (20 μg) run in a 1.2 % denaturing agarose gel was transferred to Zeta probe nylon membrane (Bio-Rad) and immobilized by UV cross-linking. Hybridization of membrane was done with random-primed α-[³²P]-labeled TSG cDNA probes as described previously [38 (link)].