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RNA extraction protocol (GNTCphenol method)

By Jason Rogers,2014-04-14 16:26
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RNA extraction protocol (GNTCphenol method)

RNA extraction protocol (GNTC/phenol method)

    (modification of Akop Seksanyan’s protocol, which in itself is a modification of Chomczynski and Sacchi’s protocol)

An RNase-free environment is critical:

    - Always wear clean gloves (don’t touch your face/hair or

    “dirty” surfaces with your gloves and assume they remain

    nuclease-free; you exude nucleases).

    - Use filter tips and other RNase-free consumables.

    - Make sure the pipette barrel is also nuclease-free.

    - Use DEPC-treated water at all times.

    Reagents (ensure sufficient volumes of all reagents are prepared in advance):

    - GNTC (NB: Add 8µl of ;-mercaptoethanol to every 1ml of

    GNTC solution before use!)

    - 2M Sodium acetate, pH 4

    - DEPC-phenol (RNase-free phenol equilibrated in DEPC-water)

    - Chloroform:Iso-amyl alcohol (49:1)

Considerations

    - Use at least 10µl of GNTC solution for every milligram of

    tissue.

    - Homogenize the sample in GNTC as soon as possible; do not

    freeze sample prior to homogenization as the nucleases

    may be active before the GNTC/;-mercaptoethanol solution

    has the chance to inhibit nuclease activity.

    - It may be necessary to use a larger volume to homogenize

    if using the Polytron. (1ml works well in a small 5ml

    snap-cap tube.)

Day One

    1. After homogenizing the tissue in the appropriate amount

    of GNTC/;-mercaptoethanol solution, add the following (in

    the order written) and vortex between each addition:

    a. 0.1V of 2M Sodium Acetate, pH 4

    b. 1V of DECP-phenol

    c. 0.3V of Chloroform:Iso-amyl alcohol (the solution

    should turn milky by the end)

    2. Incubate the mix on ice for 20 minutes; two layers should

    form.

    3. While waiting for the layers to separate, ensure the

    centrifuge is at 4ºC by doing a “fast-cool” spin. Also

    prepare collection tubes for the next step.

    4. Centrifuge at 14,000 rpm (~20,000 x g) for 25-30 minutes

    at 4ºC.

    5. Carefully transfer the top (aqueous) layer into the new

    tubes. Be VERY careful at this step not to pipet over any

    of the interphase. If necessary, only take 70-80% of the

    estimated aqueous layer.

    6. Add 1.1V of isopropanol (2-propanol) to each tube.

    7. Incubate overnight at -20ºC to precipitate. (If

    absolutely necessary, precipitate at -80ºC for 2 hours.

    Do not allow the sample to freeze.)

Day Two

    8. Again, ensure the centrifuge is at 4ºC by doing a “fast-

    spin”.

    9. Centrifuge the samples at 14,000 rpm for 25-30 minutes.

    10. Discard the supernatant very carefully. Depending on the

    tube, the pellet may be very loose. Work quickly by

    tipping most of the solution out, and follow-up with a

    quick spin, then remove the remnant with a fine gel-

    loading tip.

    11. Add 70% ethanol (use the same volume as of the

    isopropanol from the previous step).

    12. Centrifuge at 14,000 rpm for 25-30 minutes at 4ºC.

    13. As before, work quickly to remove the supernatant. Follow

    up with a second spin and carefully remove the remnant

    with a fine tip.

    14. Add 35µl of nuclease-free water.

    NB: If sufficient care was taken during pipetting of the aqueous phase, and the interphase was completely avoided, there should be no DNA in the sample. However, when extracting RNA from very small samples, the interphase is not always clearly visible, and thus carry-over of DNA often occurs. Should that happen, treatment with VERY clean DNAse can cure all known evils (OK, it can't, but it helps). We use Turbo DNase from Ambion, and have not yet had any problems. Of course, one should always check to see if one has DNA contamination in the first place, for example by doing a regular PCR (no RT!) using the RNA as a template, or by running it on a gel/RNA analysis chip.

Optional DNAseI treatment

    15. To treat with DNaseI (Turbo-DNase, Ambion), add 4µl of

    DNase buffer and 1µl of DNaseI.

    16. Incubate at 37ºC for 45 minutes to one hour.

    17. At this point, either use the DNase-inhibitor resin 1included in the kit OR repeat the extraction by adding

    (don’t forget to vortex between each addition):

    a. 500µl GNTC/;-mercaptoethanol

    b. 50µl Sodium Acetate

    c. 500µl DEPC-phenol

    d. 150µl Chloroform:Iso-amyl alcohol

     1 The resin works well enough. Add 0.1V of thawed resin (or as directed in the kit) to the sample and mix a

    few times over the course of 2 min at room temperature. Spin at max speed to pellet the resin and remove

    the RNA into a fresh tube. If using the resin, it’s easier to do the DNase treatment in 0.5ml tubes as it’s

    easier to see the resin pellet after centrifugation.

    18. Incubate for 20 minutes on ice to allow layers to

    separate. Check the centrifuge is at 4ºC and prepare

    tubes for the next step.

    19. Centrifuge at 14,000 rpm for 25-30 minutes at 4ºC.

    20. Transfer the top (aqueous) layer into fresh tubes.

    21. Add 1.1V isopropanol.

    22. Incubate at -20ºC overnight to precipitate.

Day Three

    23. Again, ensure the centrifuge is at 4ºC by doing a “fast-

    spin”.

    24. Centrifuge the samples at 14,000 rpm for 25-30 minutes.

    25. Discard the supernatant very carefully. Depending on the

    tube, the pellet may be very loose. Work quickly by

    tipping most of the solution out, and follow-up with a

    quick spin, then remove the remnant with a fine gel-

    loading tip.

    26. Add 70% ethanol (use the same volume as of the

    isopropanol from the previous step).

    27. Centrifuge at 14,000 rpm for 25-30 minutes at 4ºC.

    28. As before, work quickly to remove the supernatant. Follow

    up with a second spin and carefully remove the remnant

    with a fine tip.

    29. Add nuclease-free water.

    GNTC recipe (weights and volumes for 150ml given below) 4M Guanidine thiocyanate (70.9g / 150ml)

    25mM Sodium citrate (1.1g / 150ml)

    0.5% Sodium lauryl sarcosinate (2.5ml / 150ml)

    -pH to 7.0, filter-sterilize and store at 4ºC.

    *And don’t forget to add 8l of ;-mercaptoethanol for every 1ml of GNTC just before using it. (;-mercaptoethanol is light-

    sensitive.)

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