Alternative Protocol for Extraction of RNA from Cells Captured on LeukoLOCK™ Filters Using TRI Reagent

Description and Procedure Overview

This protocol uses TRI Reagent and silica filters for isolation of total RNA from leukocytes captured on LeukoLOCK™ leukocyte depletion filters (Ambion Cat #1933). Adjusting the amount of ethanol used to bind the RNA to the silica filter, determines whether total RNA that includes the small RNA fraction, or RNA that is depleted of smaller species such as microRNA (miRNA), tRNA, and 5S/5.8SrRNA is recovered. The quality and purity of the RNA recovered using this protocol is similar to that seen using the LeukoLOCK Total RNA Isolation System (Cat #1923), and the yields are generally higher by ~50%–100%. Microarray experiments have not been carried out using RNA extracted with this protocol, but it is not expected that alterations in the RNA profiles will occur provided the captured leukocytes are treated with RNA later immediately after filtration of the blood.

Reagents/Supplies

• TRI Reagent (e.g., Ambion Cat #9738)
• LeukoLOCK Fractionation & Stabilization Kit, Ambion Cat #1933
• Denaturing Lysis Solution, Ambion Cat #8540G (100 ml)
• Nuclease-free Water (e.g., Ambion Cat #9938, 9930)
• Nuclease-free 0.1 mM EDTA (e.g., Ambion Cat #9912, 50 ml)
• 5 M sodium chloride (e.g., Ambion Cat #9759, 9760G)
• Bromo-3-chloro-propane (BCP) (e.g., Sigma Cat #B-9673 or MRC Cat #BP151)
• 100% ethanol, ACS reagent grade or equivalent purity
• Spin Cartridges, Ambion Cat #10051G (for 50);
• Elution Tubes, Ambion Cat #12480 (for 100)
• 15 ml disposable conical plastic tubes with caps (e.g., Ambion Cat #12500)
• Nuclease-free micropipettor tips and microcentrifuge tubes (e.g., Ambion Cat #12400, 12648)
• (optional) DNAfree™ Kit, Ambion Cat #1906 (50 rxns)

Other supplies

• Blood collection tubes with EDTA as an anticoagulant; we recommend 9 ml Vacuette EDTA K3 from Greiner Bio-One #455036 or Venosafe EDTA K3 from Terumo #VF-109SDK. These tubes have lids with thin septums that are more easily pierced than stopper-type tube closures.
• 10 ml evacuated blood collection tubes, to provide a vacuum source and act as receiver tube for LeukoLOCK Filter flow-through; 10 ml Vacutainer BD #366430 or Terumo Venoject #VT-100SP recommended
• 25 G x 5/8 in hypodermic needles (e.g., BD #305122), and ~16 G hypodermic needles
• 5 ml syringes
• Disposable plastic pipets (5 or 10 ml) and bulb or mechanical pipettor

1. Collect blood samples and capture leukocytes on the LeukoLOCK Filter

Collect blood samples, and capture leukocytes on the LeukoLOCK Filter according to the LeukoLOCK Total RNA Isolation System protocol. Complete steps 1–3 in section II.B. Sample Collection, and Capture and Stabilization of Leukocytes. (You can download the protocol from our website at: http://www.ambion.com/catalog/CatNum.php?1923.)

A video of this process is available on the CD provided with the LeukoLOCK Fractionation & Stabilization Kit (Cat #1933).


2. (Optional) Flush filter with 3 ml of PBS
   
   To eliminate red blood cells and reticulocytes trapped on the LeukoLOCK Filter, flush with 3 ml of 1 X PBS according to step II.B.4 of the LeukoLOCK Total RNA Isolation System protocol.
   
   


3. (Recommended) Flush filter with 3 ml of RNAlater
   
   To stabilize the RNA in the captured leukocytes, flush the filter with 3 ml of RNA later according to step II.B.4 of the LeukoLOCK Total RNA Isolation System protocol. Do not expel the residual RNA later from the LeukoLOCK Filter, and do not retract the plunger of the syringe before detaching the syringe.


4. Seal the LeukoLOCK Filter ports
   
   Seal the LeukoLOCK Filter ports with the sheath and screw cap from the Transfer Spike.
   
   STOPPING POINT Store the LeukoLOCK Filter with stabilized leukocytes at room temperature for up to 3 days, or at –20°C or –80°C for long term storage (several months).

1. Remove RNAlater from the LeukoLOCK filter
   
   If the LeukoLOCK filter with captured cells was stored frozen, allow it to thaw at room temperature for ~5 min before proceeding.
   
   Remove the closures from the LeukoLOCK Filter. Attach a 3 or 5 ml syringe, with the plunger retracted, to the inlet port of the LeukoLOCK Filter, then depress the plunger to expel the RNAlater from the filter and ports.
   
   Typically, ~8–10 drops of RNAlater are expelled from the LeukoLOCK Filter.
   



2. Flush the LeukoLOCK Filter with 4 ml of TRI Reagent; collect lysate in a 15 ml tube
   
   In this step, the leukocytes that are trapped on the LeukoLOCK Filter are lysed, and the lysate is flushed off the filter and collected in a 15 ml conical tube.
   
   
   • Load a 5 ml syringe with 4 ml of TRI Reagent. A large-gauge needle may be used to withdraw the TRI Reagent; remove it from the syringe before proceeding to step below.
   • Attach the syringe with TRI Reagent to the inlet (flared) port of the LeukoLOCK filter, and depress the plunger of the syringe to flush the TRI Reagent through the filter, collecting the lysate in a 15 ml conical tube. The process should take 5–10 seconds. Remove the syringe, retract the plunger, re-attach it to the filter, and depress the plunger to expel residual sample trapped in the filter disk into the 15 ml tube.
   
   


3. Add 800 μl BCP, shake vigorously for 30 sec, and leave the mixture at room temp for 5 min
   
   
   • Add 800 μl BCP to the lysate and close the tube. (Alternatively, you can add the BCP to the 15 ml tube before collecting the lysate in step 2.b.)
   • Cap the tube tightly and shake the prep vigorously for 30 sec.
   • Store the sample for 5 min at room temp.


4. Centrifuge for 10 min at ~2,000 x g and recover the aqueous phase
   
   
   • Centrifuge the prep for 10 min at ~2,000 x g. (Note, this corresponds to 3,200 rpm in a Straight8 or IEC tabletop centrifuge.)
   • Remove the aqueous phase (top phase) to a fresh 15 ml conical tube. Expected volume is ~2.5–2.6 ml.


5. Add water/ethanol for isolation of total RNA or RNA depleted of small RNAs
   
   To isolate total RNA including the small RNA fraction:
   
   Measure the aqueous phase volume (use calibration marks on 15 ml tube) and add 0.5 volumes nuclease-free water and mix well. Then add 1.25 volumes 100% ethanol and mix again. For example, if the sample volume is 2.6 ml, add 1.3 ml water, mix well, then add 4.9 ml ethanol and mix thoroughly again. The prep should be homogeneous, and clear or only slightly turbid. To isolate total RNA using conditions that do not efficiently recover the small RNA fraction: Measure the aqueous phase volume and add 0.5 volumes 100% ethanol and mix well. The prep should be clear.
   
   


6. Filter sample through a Spin Cartridge
   
   Filter the sample through a Spin Cartridge (Cat #10051G) using vacuum pressure or centrifugation. Because of the large sample volume relative to the capacity of the Spin Cartridge (~700 μl), it is easier to use vacuum pressure; instructions for both methods are provided below.
   
   Vacuum filtration:
   
   
   • For each Spin Cartridge, attach a 5 ml syringe barrel to an inlet port on a vacuum manifold.
   • Insert a Spin Cartridge into the syringe barrel and apply the vacuum.
   • Use a 5–10 ml pipet to add the preparation to the Spin Cartridge, slowly adding more as the sample is pulled through the filter. Filtering the entire prep typically takes 1–2 min.
   
   Centrifugation: If a vacuum manifold/vacuum system is not available, insert the Spin Cartridge into an Elution Tube and pipet ~700 μl of sample into the Spin Cartridge. Centrifuge briefly (~10 sec) to filter the sample and discard the flow-through. Repeat to filter the entire sample.
   
   


7. Wash the Spin Cartridge with 750 μl of Wash 1
   
   
   • Place the Spin Cartridge in an Elution Tube, and add 750 μl of Wash 1.
   • Centrifuge the Spin Cartridge assembly in a microcentrifuge set to maximum speed for ~5–10 sec or until all of the solution is through the filter. (The solution will typically pass through the filter before the centrifuge reaches maximum speed.)
   • Discard the flow-through from the Elution Tube and replace the Spin Cartridge in the same Elution Tube.
   
   


8. Wash the Spin Cartridge with 2 x 750 μl of Wash 2
   
   
   • Follow the method described in the previous step to wash the Spin Cartridge with 750 μl of Wash 2.
   • Repeat with a second 750 μl of Wash 2.


9. Centrifuge the Spin Cartridge to dry the filter
   
   


10. Elute the RNA with 200–250 μl of hot 0.1 mM EDTA
    
    
    • Place the Spin Cartridge into a fresh Elution Tube.
    • Add 200–250 μl of nuclease-free 0.1 mM EDTA (preheated to 80°C) to the center of the filter in the Spin Cartridge, close the lid, and store at room temperature for 1 min.
    • Centrifuge for 1 min at maximum speed. The RNA will be in the flow-through at the bottom of the Elution Tube. Discard the Spin Cartridge. Store the eluted RNA at –20°C or –80°C.
    
    


11. (Optional) Treat eluted RNA with DNase
    
    (Optional) If desired, treat the eluted RNA with DNase to remove contaminating DNA. We recommend using Ambion’s DNAfree ™ Kit (Cat #1906), for thorough removal of genomic DNA and simplified inactivation of the DNase.
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