RNA Extraction Protocol

An RNase-free environment is critical:

* Always wear clean gloves (do not 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):


* 4M guanidine thiocyanate solution (GNTC, see recipe at bottom of page)

NB: Add 8μl of β-mercaptoethanol to every 1ml of GNTC solution before use!
* 2M Sodium acetate, pH 4
* DEPC-phenol (RNase-free acidified 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/β-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/β-mercaptoethanol solution, add the following (in the order written) and vortex between each addition:

* 0.1V of 2M Sodium Acetate, pH 4
* 1V of DECP-phenol
* 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 4 C 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 4 C.
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 -20C to precipitate. (If absolutely necessary, precipitate at -80C for 2 hours. Do not allow the sample to freeze.)

Day Two


8. Again, ensure the centrifuge is at 4C 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 4C.
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 nuclease-free water (35μl if proceeding to DNase treatment).

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 canīt, but it helps). We use Turbo DNA-free from Ambion (#1907), 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 37C for 20 to 30 minutes.

At this point, you can either use the DNase-inhibitor resin included in the kit, which takes all of 5 minutes, to remove the DNase, or you can repeat the phenol-chloroform extraction, which takes another day and a half.

Guanidine thiocyanate solution

(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 4 C.



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