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 and reviewed by the authors again in 2006  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: (stored <3 months at room temperature)
 * 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

Working solution from stock: (store <1 month at RT)
 * add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution

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