Both nuclear RNA and cytoplasmic RNA were extracted from MICs using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek) according to the manufacturer’s instructions. Total RNA was isolated using TRIzol Reagent (Invitrogen), and cDNA was obtained by reverse transcriptional RNA using FastQuant RT Kit (Tiangen), which includes DNase treatment of RNA to eliminate genomic contamination. The nuclear and cytosolic fractions were separated using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek) according to the manufacturer’s instructions. Gene expression was measured by using SYBR Premix Ex TaqTM (Takara). The small RNA was extracted by using miRcute miRNA Isolation Kit (Tiangen), and miRcute miRNA FirstStrand cDNA Synthesis Kit (Tiangen) was applied to reverse transcription of miRNAs. The expression analysis of miR-27c-3p was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen). Quantitative real-time PCR was performed in an Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific). GAPDH and 5.8S rRNA were used as negative controls to detect mRNA, lncRNA, and miRNA expression, respectively (34 ). Primer sequences are displayed in Table S1 .
Cytoplasmic nuclear rna purification kit
Manufactured by Norgen Biotek
Sourced in Canada, United States
The Cytoplasmic & Nuclear RNA Purification Kit is a lab equipment product designed for the isolation and purification of cytoplasmic and nuclear RNA from various cell types. The kit utilizes a simple and efficient process to separate the cytoplasmic and nuclear fractions, allowing for the targeted extraction of RNA from these distinct cellular compartments.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
Fastquant rt kit, by Tiangen Biotech (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Applied biosystems quantstudio 3, by Thermo Fisher Scientific (5 mentions)
Primescript rt reagent kit, by Takara Bio (5 mentions)
Mircute mirna qpcr detection kit, by Tiangen Biotech (4 mentions)
Sybr premix ex taqtm, by Takara Bio (4 mentions)
Mircute mirna isolation kit, by Tiangen Biotech (4 mentions)
Mircute mirna first strand cdna synthesis kit, by Tiangen Biotech (4 mentions)
Primescript rt master mix, by Takara Bio (3 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (3 mentions)
Mirna 1st strand cdna synthesis kit, by Vazyme (3 mentions)
Sybr premix ex taq, by Takara Bio (3 mentions)
Fluorescent in situ hybridization kit, by RiboBio (3 mentions)
Tb green premix ex taq, by Takara Bio (2 mentions)
Lysis buffer j, by Norgen Biotek (2 mentions)
Buffer sk, by Norgen Biotek (2 mentions)
Elution buffer e, by Norgen Biotek (2 mentions)
Wash solution a, by Norgen Biotek (2 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (2 mentions)
190 protocols using cytoplasmic nuclear rna purification kit
1
Nuclear and Cytoplasmic RNA Isolation and Analysis
2
Comprehensive RNA Fractionation and Expression Analysis
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Both nuclear RNA and cytoplasmic RNA were extracted from MIC cells using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek) according to the manufacturer’s instructions. Total RNA was isolated using TRIzol Reagent (Invitrogen), and cDNA was obtained by reverse transcriptional RNA using FastQuant RT Kit (Tiangen), which includes DNase treatment of RNA to eliminate genomic contamination. The nuclear and cytosolic fractions were separated using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek) according to the manufacturer’s instructions. Gene expression was measured by using SYBR Premix Ex TaqTM (Takara). The small RNA was extracted by using miRcute miRNA Isolation Kit (Tiangen), and miRcute miRNA FirstStrand cDNA Synthesis Kit (Tiangen) was applied to reverse transcription of miRNAs. The expression analysis of miR-217-5p was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen). Quantitative real-time PCR was performed in an Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific). GAPDH and 5.8S rRNA were used as negative controls to detect mRNA, lncRNA, and miRNA expression, respectively. Primer sequences are displayed in Table S1 .
3
RNA Isolation from Prostate Cancer Cells and Tissues
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Total RNA was isolated from DU145 and PC3 cells and PCa tissues using TRIzol reagent (15,596,026, ThermorFisher, USA) and total miRNA was isolated using PureLink miRNA Isolation Kits (K157001, ThermoFisher, USA). Total cytoplasmic RNA and total nuclear RNA were separately isolated using Cytoplasmic & Nuclear RNA Purification Kits (21,000, NORGEN, Thorold, ON, Canada, https://norgenbiotek.com/
4
Cytoplasmic and Nuclear RNA Fractionation
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Cytoplasmic & Nuclear RNA Purification Kits (Norgen Biotek, Canada) were used to detect ZNF667-AS1 localization in cells. The RNA of nucleus and cytoplasm was extracted, and cells were lysed using lysis buffer J and centrifuged. The precipitated pellets were added into adsorption columns. RNA was incubated at room temperature for 3 h with buffer and ethanol, and washed with hypotonic buffer to remove impurities. Then the RNA was isolated using Elution Buffer E by a 30-min incubation and centrifuged for 30 min at 15,000 ×g to separate nucleus and cytoplasm (both at 4°C). U6 was applied as a reference for nuclear RNA detection, and GAPDH was as a reference for cytoplasmic RNA detection.
5
Nuclear/Cytoplasmic Fractionation of MuSCs
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Nuclear/cytoplasmic fractionation of MuSCs was performed using a cytoplasmic/nuclear RNA purification kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions. Briefly, MuSCs (seeded at ~2 × 107 cells per dish) cultured on a 10-cm culture dish were washed twice using cold PBS after 3 days of differentiation and were lysed in a hypotonic buffer (10 mM Tris pH 8.0, 1 mM EDTA). To separate nuclei and debris, cells were centrifuged at 500× g for 5 min. Total RNA was extracted separately from the supernatant (cytosolic fraction) and the pellet (nuclei) using RNAiso Plus reagent (TaKaRa, Dalian, China) and reverse-transcribed into cDNA using the PrimeScriptTM RT Reagent Kit and gDNA Eraser (Takara, Dalian, China). RNA was quantified by qRT-PCR. 18S ribosomal RNA (18S rRNA) and U6 were used as cytoplasmic and nuclear controls, respectively. All procedures were performed at 4 °C under RNase-free conditions.
6
Comprehensive Nucleic Acid Extraction
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Genomic DNA was purified using a Blood Mini Kit (Qiagen). Total RNA was isolated using the TRIzol reagent (Invitrogen). Fractionation and extraction of small and large RNAs were performed using the NucleoSpin miRNA system (Macherey-Nagel). Separation and purification of cytoplasmic and nuclear RNAs were carried out using a Cytoplasmic & Nuclear RNA purification kit (Norgen). All the purification steps were processed according to the manufacturers’ instructions.
7
Comprehensive Tissue RNA Extraction
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Whole cell RNA preparations were carried out using TRIzol reagent (Thermo Fisher, catalog no. 15596) according to the vendor’s recommended protocol. For tumor and normal tissue, pieces were first ground finely using a mortar and pestle in liquid nitrogen prior to disruption with TRIzol reagent. Nuclear and cytoplasmic RNAs were isolated using the Cytoplasmic & Nuclear RNA Purification Kit from Norgen Biotek Corp. (catalog no. 21000) according to the vendor’s protocol. All RNA preparations were subjected to DNase treatment twice using the DNA-free kit (Thermo Fisher, catalog no. AM1906).
8
LINC00958 Subcellular Localization Analysis
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The cytoplasmic and nuclear fractions of LUAD cells were prepared using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen, CA, USA), following the manufacturer’s instructions. The expression levels of LINC00958, GAPDH, and U6 in the cytoplasmic and nuclear fractions were determined using qRT-PCR. GAPDH and U6 served as internal controls for cytoplasmic and nuclear RNAs, respectively.
9
Separation and Quantification of Cytoplasmic and Nuclear RNAs
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RNAs from the cytoplasm and nuclei were extracted separately with a Cytoplasmic Nuclear RNA Purification kit (Norgen Biotek, Thorold, Canada) according to the manufacturer’s instructions, then reverse-transcribed into cDNA with MMLV Reverse Transcriptase (Takara, Dalian, China), and QRT-PCR using TB Green® Premix Ex Taq™ (Takara, Dalian, China). The relative levels of expression were compared using the 2−ΔΔCt method.
10
Subcellular Fractionation and ncRNA Analysis
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Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA, USA) was used to isolate nuclear and cytoplasmic RNA fractions. A-375 and M21 cells were treated in cell fractionation buffer, and then subjected to centrifugation. The expression of NCK1-AS1 was detected in nuclear and cytoplasmic RNA fractions via RT-qPCR. U6 and GAPDH were taken as controls. Over three independent repeats were conducted in this assay.
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