Notes:

• Add 100 ml (for 10 Preps) or 180 ml (for 50 Preps) of 98-100% ethanol into WS Buffer bottle when first open.
• All centrifugation is at 2,500 x g (about 3,000 rpm) in a swing-bucket centrifuge.
• All plastic ware and containers should be treated properly to make sure RNase-free. Gloves should be worn when handling RNA.
• Buffers provided in this system contain irritants. Appropriate safety apparels such as gloves and lab coat should be worn.
• Pipet a required volume of RX Buffer into another tube and add 10 μl β-mercaptoethanol (β-ME) per 1 ml of RX Buffer before use.
• Some genomic DNA (add plasmid DNA, if any) will also be copurified with RNA. DNase treatment is therefore required when DNA-free RNA is desired. DNase can then be removed by phenol/chloroform extraction (refer to Protocol for “Removal of genomic DNA in eluted total RNA by DNase”).
• Complete disruption and homogenization of sample is essential for total RNA extraction.
  
  

Animal Tissues Protocol:

• Add 3.5 ml of RX Buffer (β-ME added) to 100-200 mg of liquid-nitrogen-frozen tissue, disrupt and homogenize the sample by grinding and shearing using 20-G needle-syringe. Add 10 μl β-mercaptoethanol (β-ME) per 1 ml of RX Buffer.
  
  
• Centrifuge lysate for 3 minutes to spin down insoluble material and use only the supernatant in following steps.
  
  
• Determine the final volume of the supernatant. Add an equal volume of 70% ethanol to the clear lysate and mix by vortexing. If lysate is lost during the preparation, reduce the volume of ethanol accordingly. Do not centrifuge the ethanol added lysate.
  
  
• Place a Total RNA Midi Column onto a 15 ml Collection tube. Add 5 ml of the ethanol-added sample (including any precipitate) into the column. Centrifuge for 3 minutes and discard the flow-through. Repeat this step for the rest of the sample. If some sample still remains in the column, repeat centrifugation until all sample pass the column.
  
  
• Wash the column once with 5 ml of WF Buffer by centrifuging for 30-60 seconds, discard the flow-through.
  
  
• Wash the column twice with 7 ml of WS Buffer by centrifuging for 30-60 seconds, discard the flow-through. Add 100 ml (for 10 Preps) or 180 ml (for 50 Preps) of 98-100% ethanol into WS Buffer bottle when first open.
  
  
• Centrifuge the column for an additional 5 minutes to remove ethanol residue.
  
  
• Place the column onto a new 15 ml tube. Add 0.5 ml of RNase-free ddH2O (provided) onto the center of the membrane. For effective elution, make sure that the elution solution is dispensed onto the center of the membrane.
  
  
• Stand the column for 2 minutes, and centrifuge for 5 minutes to elute total RNA.
  
  
• Store RNA at -70º C.
  
  

Animal Cells protocol:

• Harvest 3 x 107 - 7 x 107 cells and centrifuge at 300 x g for 5 minutes to pellet cells. Remove all the supernatant.
  
  
• Disrupt cells by adding 3.5 ml (3 x 107 cells), 7 ml (7 x 107 cells) of RX Buffer (β-ME added), and vortexing the sample. Homogenize sample by using 20-G needle-syringe. Add 10 μl β-mercaptoethanol (β-ME) per 1 ml of RX Buffer.
  
  
• Follow the Animal Tissues Protocol starting from Step 2.
  
  

• Prepare cytoplasm lysate. Prepare cell lysis buffer (provide by user): 20 mM Tris-HCl pH 8.0, 1 mM MgCl2, 0.5% NP-40.Keep at 4º C.Only fresh cells are used for preparing cytoplasm lysate.
• Harvest 3 x 107 -7 x 107 cells and centrifuge at 300 x g to pellet cells.
• Add 1.8 ml of cell lysis buffer to cell pellet, resuspend and lysis cells by gentle pipetting, and incubate on ice for 5 minutes.
• Centrifuge the lysate at 300 x g at 4°C for 3 minutes, transfer the supernatant to a new tube, and use the supernatant (lysate) in following steps.
  
  
• Add 6 ml of RX Buffer (β-ME added) to the lysate and mix by vortexing. Add 10 μl β-mercaptoethanol (β-ME) per 1 ml of RX Buffer.
  
  
• Add 4.5 ml of 98% ethanol to the sample and mix by vortexing.
  
  
• Follow the Animal Tissue Protocol starting from Step 4.
  
  

• Pellet up to 1 x 1010 bacterial cells by centrifuging at 2,500 x g (3,000 rpm) for 5 minutes to pellet cells.
  
  
• Resuspend cells in 1 ml of TE buffer by vortexing.
  
  
• Add lysozyme (provide by user) to final concentration of 500 μg/ml for Gram-negative bacteria; 2 mg/ml for Gram-positive bacteria, and incubate at room temperature for 10 minutes.
  
  
• Add 3.5 ml of RX Buffer (β-ME added) to the sample and vortex. Add 10 μl β-mercaptoethanol (β-ME) per 1 ml of RX Buffer.
  
  
• Centrifuge lysate for 3 minutes to spin down insoluble material and use only the supernatant in the following steps.
  
  
• Add 2.5 ml of 98% ethanol to the sample and mix by vortexing.
  
  
• Follow the Animal Tissue Protocol starting from Step 4.
  
  

Troubleshooting:

• Insufficient disruption or homogenization.
  Reduce the amount of starting sample and perform more disruption and homogenization.
  
  
• Clogged Total RNA column. Reduce the amount of starting sample and perform more disruption and homogenization. Centrifuge the lysate to remove insoluble materials and use the supernatant only.
  
  
• RNA is degraded. Starting sample should be fresh or frozen in liquid nitrogen and store at -80oC. Improper handling of the sample or storing the sample at -20 oC will cause the RNA degradation.
  
  
• RNase contamination. Use RNase-free liquid, handling tips and tubes.
  
  

• Use ddH2O of acidic pH to dilute RNA sample for spectrophotometric analysis.
  Use 10 mM Tris-HCl of pH 7.5 or TE buffer to dilute RNA sample.
  
  
• DNA is copurified with RNA
  Refer to Protocol for “Removal of genomic DNA in eluted total RNA by DNase”.