Protocol for Determining RNA Localization by Subcellular RNA Fractionation

Irfana Saleem^1, 2, $, Bhopal C. Mohapatra^1, 4, Hamid Band^{1, 2, 3, 4, 5}, Vimla Band^{1, 2, 4, #}

¹ Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, USA.

² Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

³ Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

⁴ Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.

⁵ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center Omaha, Nebraska, USA.

^$ Current Address: Therapeutic Innovation Center, Baylor College of Medicine, Houston, Texas, USA

^# For Correspondence Email: vband@unmc.edu

This protocol is intended to extract nuclear and cytoplasmic RNA from cultured cells and total RNA from whole cells. # This protocol is adapted from https://www.activemotif.com/catalog/1160/rna-subcellular-isolation-kit

Materials required.

• 1 X PBS (Gibco, cat#10010023) warmed at 37^oC.
• Culture media for cells (based on the cells’ requirement)
• Trypsin-EDTA (Gibco, cat#15400-054)
• Trypsin Inhibitor (Roche, cat# 10109886001)
• RNA isolation kit cat # 25501 (Active Motif Inc, cat # 25501)

# Desired number of total cells required is at least 6 x 10⁶ but should be adapted for experimental requirements.

• Seed 3 x 10⁶ cells in each 10 cm dish 24h prior to the experiment.
• After 24 h, wash the cells with 1 X PBS
• Add 2 ml of 0.05 % trypsin-EDTA to the plates and incubate at 37⁰C for -1-5 minutes based on the cell types.
• Neutralize the trypsin with 2 ml of trypsin inhibitor.
• Collect and combine the cells from three plates into single 15 ml conical falcon tube and centrifuge the cells for 5 min at 1000 rpm.
• After centrifugation remove the supernatant medium and resuspend the cells in 5 ml of cell culture medium.
• Count the cells using a hemacytometer or Countess II FL automated cell counter (ThermoFisher Scientific) and Trypan blue.
•  Take approximately 6 X 10⁶ cells into a different conical tube and repeat the centrifugation for 5 min at 1000 rpm.
• After the centrifugation, remove the medium and resuspend the cells into 2 ml of 1 x PBS and split them into equal half by taking 1 ml of cells in PBS into 1.5 ml microcentrifuge tubes label #1 and #2.
  #1 is for direct extraction of total RNA from cells and # 2 is for the subcellular RNA fractionation.
• Centrifuge them at 14,000 rpm for 5 min at 4⁰C and remove 1 X PBS from both the tubes and proceed for RNA extraction based on the instructions below or manufacturer’s protocol.
  
  # Please refer to buffer composition from the manufacturer’s instructions and make all the required buffer prior to this step

Total RNA isolation

• Resuspend the cell pellet from tube #1 with 350 µl Complete Buffer G for 1x reaction.
• Vortex tubes on highest setting for 30 seconds
• Add 350 µl of 70% ethanol to each tube.
• Transfer this to the column specified by the kit and continue the centrifugation and perform wash steps and elute RNAs in nuclease free water as specified in the manufacturer's instructions specified in https://www.activemotif.com/documents/2021.pdf

• Store the RNA in 4⁰C for short term like within hours or store it in -80⁰C for long term uses.

   

Cytoplasmic-Nuclear RNA fractionation

• Resuspend each cell pellet from tube #2 in 120 µl Complete Lysis buffer.
• Incubate for 10 minutes on ice.
• Centrifuge at 14,000 rpm for 5 minutes at 4°C.
• Prepare a new RNase-free microcentrifuge tube for each sample and label as cytoplasmic. Transfer the supernatant to the new tube labelled as cytoplasmic. Take care to remove all the supernatant without disturbing the pellet.
• The remaining pellet contains nuclear fraction. Label the tubes as nuclear. Wash the nuclear pellets by adding 150 µl of 70% ethanol to each tube. Centrifuge at 14,000 rpm for 5 minutes at 4°C. Carefully remove and discard supernatant.
• Add Complete Buffer G to each tube as follows: 
  Cytoplasmic: Add 250 µl Complete Buffer G 
  Nuclear: Add 350 µl Complete Buffer G
• Vortex all tubes on highest setting for 30 seconds.
• Add 350 µl of 70% ethanol to all tube.
• Homogenize the pellets by continuous pipetting.
• Prepare separate purification columns for each cytoplasmic and nuclear sample.
  Add the entire sample to its corresponding purification column and continue the centrifugation and perform wash steps and elute RNAs in nuclease free water as specified in the manufacturer's instructions specified in https://www.activemotif.com/documents/2021.pdf
   

Measuring subcellular localization with qRT-PCR

# cDNA preparation and primers are to be optimized for the experimental requirements.

• Take 1 µg of RNA from total cellular RNA, cytoplasmic RNA and nuclear fractionated RNA and add random hexamer for reverser transcription using SuperScript II reverse transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Detailed protocol for RT is linked below.
  https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFSAssets%2FLSG%2Fmanuals%2FsuperscriptII_pps.pdf

• After RT, the cDNA was diluted to 1 :5 ratio to input into aliquots and were used for quantitative real-time PCR (qRT-PCR) analyses with specific primer sets (Sigma, St. Louis, MO) listed below. qRT-PCR was carried out in an Applied Biosystems 7500 real-time PCR system using Power SYBR green master mix from Applied Biosystems (Thermo Fisher Scientific, cat# 4368577) using specific primers.

   

Data analysis and normalization

• Primers for nuclear and cytoplasmic localizing RNAs were used as controls to confirm the subcellular RNA fractionation.
• The RNAs from nuclear or cytoplasmic RNAs were normalized to corresponding total cellular RNA and fold change was calculated.

% of Input = 100 x [2^ (Ct total RNA – Ct RNA fraction)]

This is also discussed in detail in https://www.activemotif.com/documents/2021.pdf

• Reference gene specific to the cellular compartment also can be used to normalize the gene of interest.
  Fold change = 2^ (Ct control gene – Ct test gene)
  This is also discussed in detail in https://www.activemotif.com/documents/2021.pdf

References:

Saleem I, Mirza S, Sarkar A, Raza M, Mohapatra B, Mushtaq I, Kim JH, Mishra NK, Alsaleem MA, Rakha EA, Qiu F, Guda C, Band H, Band V. The Mammalian Ecdysoneless Protein Interacts with RNA Helicase DDX39A To Regulate Nuclear mRNA Export. Mol Cell Biol. 2021 Jun 23;41(7):e0010321. PMID: 33941617; PMCID: PMC8224239.