Introduction

With a few additional reagents and significant protocol modifications, the RiboPure™-Blood Kit (Cat #AM1928) can be used to isolate total RNA that includes the small RNA fraction from RNA later® Solution-treated human blood samples. Using this modified protocol, total RNA that includes miRNA, tRNA, 5S/5.8SrRNA, and other small RNA species is recovered.
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Materials

 
  • RiboPure™-Blood Kit, Ambion Cat #AM1928
  • Acetic Acid, glacial or >99.5%, ACS reagent grade or equivalent
  • 100% Ethanol, ACS reagent grade or equivalent
  • Denaturation Solution, Ambion Cat #AM8540G
  • Wash solutions

For this protocol, prepare wash solutions following Tables 1 and 2 below; the volumes shown are enough to process 40 samples. Note that the wash solutions from the RiboPure-Blood Kit cannot be used for this modified
protocol.

Table 1. [70% Ethanol/30% Denaturation Solution]

Component For 35 mL
100% Ethanol (ACS reagent grade or equivalent)24.5 mL
Denaturation Solution, Ambion Cat #AM8540G10.5 mL


Table 2. [80% Ethanol/50 mM NaCl]

Component For 70 mL
100% Ethanol (ACS reagent grade or equivalent)56 mL
5M NaCl0.7 mL
Nuclease-free Water13.3 mL

Equipment and supplies

  • General laboratory equipment including microcentrifuge, pipettors, RNase-free tips, and heat block or incubator set to 80°C
  • Blood collection tubes (recommended anticoagulant: potassium EDTA or sodium EDTA)
  • 2 mL polypropylene microfuge tubes capable of withstanding 16,000 x g (e.g. 2 mL RNase-free Microfuge Tubes, Ambion Cat #AM12425)
  • 15 mL screw cap tubes
  • (Recommended) Vacuum manifold set up with 5 mL syringe barrels to support the Filter Cartridges supplied with the RiboPure-Blood Kit
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Protocol - Sample Collection, Cell Lysis, and Initial RNA Purification

  1. Collect blood and add 300–500 μL blood to 1.3 mL RNAlater® Solution

  2. Follow the instructions in section II.A on page 5 of the RiboPure-Blood protocol to collect human blood. Stabilize samples by adding 300–500 μL blood to 1.3 mL RNAlater Solution in a 2 mL microcentrifuge tube (tube is not provided with the RiboPure-Blood Kit).

  3. Centrifuge sample and discard the supernatant

    • If sample is frozen, thaw completely at room temperature.
    • Centrifuge sample for 1–2 min at maximum speed in a microcentrifuge. The blood cells and plasma proteins will form a large brown or reddish-brown pellet which may smear upward along the side of the tube, and the supernatant may be pale pink, brown, or colorless (but it is often turbid).
    • Remove and discard the supernatant by aspiration or pouring.•
    • When aspirating the supernatant, thoroughly remove all of the fluid, including the portion directly above the cell pellet, which may be more turbid, and which may contain some white particulate matter. Note, this material is not the “buffy coat” fraction seen in untreated whole blood after centrifugation.
    • If the supernatant is removed by pouring, tap the rim of the inverted tube gently against a paper towel to remove all residual fluid.
    • Remove any fluid from inside the tube cap.


  4. Add 800 μL Lysis Solution and vortex vigorously

    • Add 800 μL Lysis Solution from RiboPure-Blood Kit to the pelleted blood sample.
    • Vortex vigorously to resuspend the pellet.

  5. Add 10 μL acetic acid and store on ice for 5 min

    • Add 10 μL acetic acid and vortex to mix thoroughly.
    • Store on ice for 5 min.


  6. Extract with 500 μL Acid-Phenol: Chloroform

    • Withdraw 500 μL of Acid-Phenol:Chloroform from beneath the overlying layer of aqueous buffer, add it to the cell lysate, and shake vigorously or vortex for 30 sec.
      Proceed immediately to the next step, do NOT store the mixture for 5 min as in the RiboPure-Blood Kit protocol.

      NOTE   If addition of 500 μL of Acid-Phenol:Chloroform would cause the tube to be too full to permit adequate mixing, you can use as little as 250 μL of Acid-Phenol:Chloroform.

    • Centrifuge at room temp for 1 min at maximum speed in a microcentrifuge to separate the aqueous and organic phases. The aqueous phase may appear cloudy or clear after centrifugation.


  7. Transfer the aqueous phase to a 15 mL tube

    Transfer the aqueous (upper) phase containing the RNA to a 15 mL screw-cap tube.

    • Typically the aqueous phase volume is ~1.2 mL.

    • Avoid transferring the colored material from the organic (lower) phase, which contains heme and proteins. Discard the lower phase.


  8. Add 1 ml of Denaturation Solution and 2.7 mL of 100% ethanol

    • Add 1 ml of Denaturation Solution (Ambion Cat #AM8540G) then mix thoroughly by vortexing. At this point, the volume of the prep should be ~2.2 mL.
    • Add 2.7 ml of 100% ethanol (i.e., ~1.25 volumes of ethanol with respect to the prep volume). At this point, the volume of the prep should be ~5 mL.
    • Mix thoroughly by vortexing for ~3–5 sec. Observe the preparation to ensure that the solution becomes clear. If the solution remains cloudy, add nuclease-free water in 300 μL increments, mixing after each, until the solution becomes clear.
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Protocol - Final RNA Purification

Before you start, heat >100 μL per sample of Elution Solution to ~75°C in an RNase-free tube.

  1. Pass the sample through a Filter Cartridge

    Filter the sample through a Filter Cartridge using a vacuum manifold. Alternatively, sample can be centrifuged though a Filter Cartridge by successively applying 700 μL of sample, centrifuging for a few seconds to pass the liquid through, emptying the Collection Tube and repeating. Discard the filter flow-through.

  2. Wash filter with 700 μL [70% Ethanol/ 30%Denaturation

    • Remove the Filter Cartridge from the vacuum manifold and transfer it to a 2 mL Collection Tube (included with the RiboPure-Blood Kit)

    • Apply 700 μL [70% Ethanol/30% Denaturation Solution] to the Filter Cartridge and centrifuge for ~5–10 sec to pass the solution through the filter. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube.


  3. Wash filter with 2 x 700 μL [80% Ethanol/50 mM NaCl]

    • Apply 700 μL [80% Ethanol/50 mM NaCl] to the Filter Cartridge and centrifuge for ~5–10 sec to pass the solution through the filter. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube

    • Repeat with a second 700 μL aliquot of 80% Ethanol/50 mM NaCl.

    • After discarding the flow-through from the last wash, replace the Filter Cartridge in the same Collection Tube and spin the assembly for 1 min to remove residual fluid from the filter.


  4. Elute RNA with 150 μL preheated Elution Solution

    • Transfer the Filter Cartridge into a labeled Collection Tube (provided with the RiboPure-Blood Kit).

    • Apply 150 μL Elution Solution (preheated to ~80°C) to the center of the filter, and close the cap. Leave the assembly at room temp for 1 min

    • Centrifuge for ~20–30 sec at maximum speed to recover the RNA in the Collection Tube.
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LT037                 17-May-2007