Sureprep nuclear or cytoplasmic rna purification kit

SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit (Fisher BioReagents) was applied to isolate nuclear and cytoplasmic fractions according to the manufacturer’s instructions. RNA levels of lncRNA GRIK1-AS1, the nuclear control RNU6-1, and the cytoplasmic control GAPDH were analyzed by real-time qPCR.

HCT116 and LoVo cells were harvested for subcellular RNA isolation. Cell cytoplasm and nuclear fractionation was performed with SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific, MA) in accordance with provider’s manual. RNA quality and quantity were determined with Nanodrop 2000 (Invitrogen, MA). The U6 and GAPDH transcripts were employed as nuclear and cytosolic controls, respectively.

Total cellular RNA and subcellular RNA fractions were isolated from cells treated and untreated with 5-FU. Subcellular fractionation was performed with the SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit (Fisher BioReagents, Canada) according to the manufacturer’s instructions. Cells grown in 100 mm plates were rinsed twice within ice-cold PBS and lysed in 200 μL of ice-cold lysis solution. After 5 min on ice, cell lysates were centrifuged at 12,000× g for 15 min at +4 °C. The supernatant was recovered in the cytoplasmic fraction, the pellet contained nuclear RNA. Purified total, cytoplasmic and nuclear RNAs were analyzed by RT-qPCR. Relative RNA abundance was corrected according to the portion of the total RNA present in each fraction. We have analyzed the expression of LINC00973 and DCBLD2 genes in total cellular RNA and subcellular RNA fractions. NEAT1, RNU6-1 (nuclear localization) and GAPDH (cytoplasmic localization) were used as controls.

Propidium iodide, APC-Annexin V and protease inhibitor cocktail were provided by Sigma (St. Louis, MO, USA); CCK-8 test kit was provided by Dojindo Corp (Kyushu, Japan); lipofectamine 3000 and Trizol were from Thermo Fisher Scientific (Waltham, MA, USA); Apoptosis Detection Kit was from Beyotime (Nanjing, China); Dual-Luciferase Reporter Assay System was provided by Promega (Madison, WI, USA); matrigel was from BD (New Jersey, USA); Macoy’s 5A medium was from Gibco (Rockford, MD, USA); SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit was from Fisher BioReagents^® (Fair Lawn, NJ, USA). Antibodies against the Bcl-2 (# 3498), GAPDH (#2118) and Cleaved caspase3 (# 9661S) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); antibody against the DACT1 was from Novus Biologicals, LLC (Centennial, CO, USA).

Nuclear/cytoplasmic fractionation was performed as described.^{53 (link)} Briefly, cells were rinsed three times with cold phosphate-buffered saline (PBS) and scraped carefully. After spinning, disruption buffer (KCl 10 mM, MgCl₂ 1.5 mM, Tris-Cl, pH 7.5, 20 mM, and dithiothreitol (DTT) 1 mM) was added and let to stand for 10 min. A type B Dounce was used to disrupt the cytoplasm using 10–15 strokes. When ~90% of the cytoplasm was broken, 0.1% of Triton X was added, mixed by inversion five times, and centrifuged for 5 min at 1500 r.p.m. The supernatant containing the cytoplasmic fraction was recovered by this method. Both supernatant and nuclei (pellet) were processed for RNA extraction using the SurePrep Nuclear or Cytoplasmic RNA Purification Kit (Fisher, BP2805-50) following user manual instructions. DNAaseI treatment was performed for both fractions (Norgen Biotek cat. num. 25710).

Both nuclear and cytoplasmic RNA from cultured DU145 or 22Rv1 cells were isolated by SurePrep Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific BP2805-25) followed manufactuere's instruction. U1 RNA and GAPDH processed mRNA were detected in isolated RNAs as control for nuclear RNA and cytoplasm RNA, respectively. Biological triplicates were carried out and followed by qRT–PCR to detect abundance of lncRNAs.