RNA extraction using self-made guanidinium-acid-phenol reagents

RNA extraction with guanidinium-acid-phenol reagents based on the research of Chomczynski P, Sacchi N. 1987 [1] and reviewed by the authors again in 2006 [2] is generally considered the extraction method that gives the best quality RNA. If you prefer using ready-made reagents like TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) refer to this protocol page: RNA extraction using trizol/tri.

Mixing your own "TRI" reagent

So called Solution D (based on Chomczynski and Sacchi 1987/2006) is:

• 4 M guanidinium thiocyanate
• 25 mM sodium citrate
• pH 7.0
• 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl)
• 0.1 M 2-mercaptoethanol

Prepare stock with:

• dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 °C
• add 17.6 ml of 0.75 M sodium citrate, pH 7.0
• 26.4 ml of 10% (wt/vol) N-laurosylsarcosine

(stored <3 months at room temperature)

Working solution from stock:

• add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution

(store <1 month at RT)

Using Solution D

Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):

• add 1 ml solution D (don't linger on this step)
• transfer to tubes
• add 0.1 ml of 2 M sodium acetate, pH 4.0, and invert tube to mix
• add 1 ml water-saturated phenol (never buffered phenol) and invert tube
• add 0.2 ml of chloroform/isoamyl alcohol (49:1) and shake vigorously for 10 sec
• centrifuge 20min 10000G 4ºC
• transfer top aqueous phase into new tube
• precipitate