• Isolation
• Quantitation
• Gel Electrophoresis

When working with RNA: always wear gloves, use only freshly prepared and autclaved material and use sterile plastic pipettes. It's a good idea to use DEPC-treated material but this is not really necessary as long as sterile equipments are used.

RNA can be isolated using various methods.

• Guanidine-based isolation
• Phenol-based isolation
• TRIZOL Reagent (Gibco BRL)
• RNA isolation kits (QIAGEN, Promega, etc.)
  

We consistantly isolate good quality RNA from PTEC using the RNeasy midi kit from QIAGEN

• Immediately after the desired incubation period, aspirate cell culture medium+stimulant. Note: cells are grown in 10 cm TC dish.
  
• Add 2 ml of RLT lysis buffer (+b-ME) directly on to the cells and collect cell lysate with a rubber policeman and pipet lysate into an Rnase-free polypropylene tube.
  
• Vortex the sample for 10 sec and pass the lysate at least 5-10 times through an 18-20 gauge needle.
  
• Add 2 ml of 70% ethanol to the homogenized lysate and mix thoroughly by shaking vigorously.
  
• Apply the sample to an RNeasy midi spin column placed in a 15-ml centrifuge tube. Centrifuge for 5 min at 3000-5000 g. Discard flow-through.
  
• Add 4 ml buffer RW1 to the column. And centrifuge as above. Discard flow-through.
  
• Add 2.5 ml Buffer RPE to the column. Centrifuge for 2 min and discard flow-through.
  
• Add another 2.5 ml Buffer RPE to the column. Centrifuge for 5 min to dry column.
  
• Transfer the column to a new 15 ml collection tube and add 150 ul of Rnase-free water directly onto the spin-column membrane. Let stand for 1 min, and centrifuge for 3 min.
  
• Store RNA in water at -70^oC.
  

RNA QUANTITATION

• Purity - pure RNA has an A260/A280 ration of 1.9-2.1 in 10 mM Tris-Cl, pH 7.5
  
• Quantitation - 1 absorbance unit at 260 nm corresponds to 40 ug of RNA per ml. This relation is valid only for measurements in water.
  
• 1 A260 = 40 ug/ml
  

Reagents

• Running Buffer - 25 ml 10x MOPS + 225 ml MQ H2O
  
• Sample loading Buffer - 10 ul 10x MOPS + 17 ul formaldehyde + 50 ul formamide + 15 ul 6x gel loading solution
  
• Agarose
  
• Ethidium Bromide
  
  

Procedure

• Mix 24 ml 1% agarose gel (60^oC) with 3 ml formaldehyde, 3ml 10x MOPs
  
• Set gel in casting tray (10x7 cm)
  
• Denature samples (10 ul RNA samples + 10 ul sample loading buffer) by incubating at 65oC for 5 min. Chill on ice.
  
• Run at 75V for approx. 45-60 min.
  
• Stain with 5 ul EtBr (10mg/ml) in 100ml dH^₂O for 1-3, wash in dH₂O for 5 min. View under UV light.
  
• Ribosomal bands should appear as sharp bands on the stained gel. 28S ribosomal RNA bands should have twice the intensity as that of the 18S RNA band. Human 18S = 1.9 kb, 28S = 5 kb.