Active Motif,
Tools to analyze nuclear function,
Your CartYour Cart 0 items
サンプル調製

RNA Subcellular Isolation Kit

核,細胞質または全RNAの分離

RNA Subcellular Isolation Kit は,交差汚染なしに核または細胞質RNAを分離するための効率的な方法を提供します。RNA分子は細胞内に常に異なる成熟とプロセシングレベルで存在しています。この不均質性は,下流解析とデータ解釈にバイアスをかけます。特定の細胞下分画からRNAを分離することによって,遺伝子発現解析の正確性は大きく改善されます。細胞内分離は,他のイントロンまたは成熟RNAからバックグラウンドを減少させることによって,低量の転写産物を特定する可能性が増進し,RNAプロセシング力学研究を援助します。さらに細胞内分離は,長鎖非翻訳RNAlncRNA)が,機能的な役割に対する重要な洞察を提供する作用部位に局在するのを助けます。

RNA Subcellular Isolation Kitには,1反応につき200万~400万の培養細胞または15mgの組織から15の核および15の細胞質精製を行うのに十分な試薬が含まれます。試料を溶解させ,遠心分離するだけで,核および細胞質RNAを分離することができます。各RNA断片を個々のスピンカラムに充填し,タンパク質を除去するために洗浄します。精製されたRNAをカラムから抽出し,定量します。 この解析は,全RNAの分離にも使用することができます。

アクティブ・モティフ社のRNA Subcellular Isolation Kitの詳細については,以下の Description, Method, Data または Contents タブをクリックしてください。 マニュアルまたは関連書類については,Documents タブをクリックしてください。

 

 
Name Format Cat No. 価格 (税抜)  
RNA Subcellular Isolation Kit 30 rxns 25501 ¥44,000 Buy Now

Kit Specifications:

  • Separates nuclear and cytoplasmic RNA without cross-contamination
  • Works with cultured cells or tissue
  • Easy-to-use spin columns included for RNA purification
  • Method avoids the use of phenolic compounds
  • Isolates RNA between the sizes of 15-7,000 nucleotides (includes miRNA, piRNA, snoRNA, mRNA, lncRNA)
  • Purified RNA validated for use in RT-qPCR and RNA-seq
Agarose gel image of nuclear and cytoplasmic RNA isolated withe the RNA Subcellular Isolation Kit
Figure 1: Agarose gel of nuclear and cytoplasmic RNA fractions.

RNA was isolated as nuclear and cytoplasmic fractions using the RNA Subcellular Isolation Kit. Following purification, half of the nuclear RNA was treated with DNase I to remove genomic DNA contamination using the DNase I Treatment Kit (Active Motif Catalog No. 25502). 1 µg of each RNA fraction was loaded onto a 1.5% gel for analysis.
Lane M = 100 bp DNA ladder
Lanes 1-2 = Cytoplasmic RNA
Lanes 3-4 = Nuclear RNA before DNase I treatment
Lanes 5-6 = Nuclear RNA after DNase I treatment

 

TapeStation trace of cytoplasmic RNA isolated with the RNA Subcellular Isolation Kit
Figure 2: TapeStation trace of cytoplasmic RNA isolated with the RNA Subcellular Isolation Kit.

Cytoplasmic RNA was isolated from HeLa cells using the RNA Subcellular Isolation Kit. TapeStation analysis was performed to analyze the recovered RNA before and after rRNA depletion. The yellow and green traces represent two cytoplasmic samples pre-RNA depletion. The two large peaks represent the 18S and 28S rRNA. The blue and red traces represent the same samples after rRNA depletion. The 18S and 28S peaks are no longer present. The size of the purified RNA spans from 15-7000 nucleotides.

 

sRNA distribution frequency between cytoplasmic and nuclear fractions
Figure 3: Small RNA distribution frequency between cytoplasmic and nuclear fractions.

Cytoplasmic and nuclear RNA were isolated from HeLa cells using the RNA Subcellular Isolation Kit. RNA was processed with the NEBNext® Multiplex Small RNA Library Prep Kit and size selected to remove large RNA. 75 bp single read sequencing was performed and the data was mapped to miRBase. A frequency of the small RNA (sRNA) distribution for each fraction shows the specificity of subcellular isolation.

 

Applications:

The RNA Subcellular Isolation Kit is designed to isolate nuclear and cytoplasmic RNA from cultured cells and fresh tissue, or total RNA from cultured cells and fresh or frozen tissue. The purified RNA is suitable for use in a variety of downstream applications.

  • Reverse-transcription quantitative PCR (RT-qPCR)
  • RNA-seq
  • Northern blotting
  • Purified RNA can undergo DNase I treatment
  • Purified RNA can undergo ribosomal RNA (rRNA) depletion
  • Purified RNA can be used for polyadenylation (poly A) selection
RT-qPCR Expression Data of nuclear and cytoplasmic RNA fractions purified with the RNA Subcellular Isolation Kit
Figure 4: Subcellular RNA Isolation to identify RNA localization.

Nuclear, cytoplasmic and total RNA were isolated from HeLa cells using the RNA Subcellular Isolation Kit. Following purification and DNase I treatment of the nuclear and total RNA fractions with Active Motif's DNase I Treatment Kit (Catalog No. 25502) purified RNA was subjected to reverse transcription and qPCR. Each subcellular fraction was plotted as a percentage of total RNA. The data shows the localization of various RNAs. MALAT1, NEAT1 and TUG1 are lncRNAs. Their expression data is consistent with published RNA-FISH data for the same RNAs and cell type. Both S14, which is a ribosomal RNA, and BIRC5, a mRNA, are expected to localize within the cytoplasm as demonstrated above.

 

RNA-Seq data of nuclear and cytoplasmic RNA fractions purified with the RNA Subcellular Isolation Kit
Figure 5: RNA-Seq data confirms lncRNA localization.

Nuclear, cytoplasmic and total RNA were isolated from HeLa cells using the RNA Subcellular Isolation Kit. RNA was subjected to ribosomal RNA depletion during NGS library preparation. Samples were then sequenced using the Illumina HiSeq and 100 bp paired end reads with 50 M reads per sample. NEAT1 is a lncRNA that is primarily located in the nucleus, while TUG1 is a lncRNA known to have both nuclear and cytoplasmic localizations (see Figure 3 above for RT-qPCR data). This data is also consistent with published RNA-FISH data and RT-qPCR analysis.

How does it work?

The RNA Subcellular Isolation assay works by lysing cell or tissue samples in complete lysis buffer. The lysate is then separated by centrifugation, with the supernatant containing cytoplasmic RNA and the pellet containing nuclear RNA. A guanidine-based buffer and ethanol are added to each RNA fraction before being loaded onto individual spin columns. Following a wash step to remove any proteins, the RNA fractions are eluted and quantified. RNA can be further process with Active Motif's DNase I Treatment Kit (Catalog No. 25502) to remove any genomic DNA contamination from the purified RNA sample prior to downstream analysis.

Flow Chart of the RNA Subcellular Isolation Kit which can efficiently purify nuclear, cytoplasmic and total RNA from cells and tissues
Figure 1: Flow Chart of the RNA Subcellular Isolation Method.

  

RNA Subcellular Isolation Kit
Maximum Column Binding Capacity  40 µg
Maximum Column Loading Volume  750 µl
Size of purified RNA  15-7,000 nucleotides
Maximum Amount of Starting Material
  Cells

  Tissue
 
  2 x 106 cells (Total RNA)
  4 x 106 cells (Cytoplasmic and Nuclear RNA)
 15 mg tissue
Assay Time  2 hours
Average Yields
  HeLa Cytoplasmic RNA
  HeLa Nuclear RNA
  HeLa Total RNA

 12-25 µg at 250-500 ng/µl from 2 x106 cells
 12-25 µg at 250-500 ng/µl from 2 x106 cells
 25-40 µg at 500-900 ng/µl from 2 x106 cells

Contents & Storage

Please note that the RNA Subcellular Isolation Kits are shipped on dry ice and contain reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.This kit includes the following components:

  • 1 M DTT; Store at -20°C
  • 10X PBS; Store at 4°C
  • Lysis Buffer AM7; Store at 4°C
  • Buffer G; Store at 4°C
  • Purification Columns; Store at RT
  • DEPC Water; Store at RT