N.B: Care must be taken when handling solutions containing high concentrations of guanidine salts due to its chaotropic nature. As with other procedures involving RNA, gloves should be worn at all times to avoid contamination of samples with ribonucleases.
This method describes preparation from small quantities (~ 50mg) of tissue. Appropriate scaling of the volumes involved can be performed to accomodate larger quantities.
You will need:
Guanidine isothiocyanate (Sigma)
1M sodium citrate, pH 7 (DEPC-treated, autoclaved)
Sarcosyl (N-lauryl sarcosine, Sigma)
b-mercaptoethanol (Sigma)
2M sodium acetate, pH4 (DEPC-treated, autoclaved)
3M sodium acetate, 100mM magnesium acetate, pH 5.2
Absolute ethanol
Propan-2-ol (isopropanol)
70% ethanol (made with DEPC-treated, autoclaved water)
0.5% SDS (made with sterile, DEPC-treated water)
Solution D - 4M guanidine isothiocyanate, 25mM sodium citrate, pH7, 0.5% sarcosyl, 100mM b-mercaptoethanol.
Solution D can be made and stored at 4°C without b-mercaptoethanol for several months. b-mercaptoethanol should be added to 100mM immediately prior to use. b-mercaptoethanol is used to prevent the reformation of the intra-molecular disulphide bridges , one of the reasons for the extreme stability of ribonucleases.
1) Tissue is homogenized as rapidly as possible, at 4°C, in solution D (500ul per 50mg tissue) with an eppendorf pestle homogenizer until a smooth, lysed, homogenous suspension is obtained.
2) Add 50ul 2M sodium acetate, pH4.0 and mix vigorously.
3) Add 500ul phenol and mix vigorously.
4) Add 100ul chloroform, mix vigorously and incubate on ice for 15 minutes.
5) Centrifuge mixture at 10,000g for 10 minutes in a microfuge at 4°C.
6) Remove upper, aqueous phase to a clean, sterile, DEPC-treated eppendorf tube. After centrifugation, RNA is present in the aqueous phase while, due to protonation at the acidic pH used, genomic DNA is partitioned into the phenol phase.
7) Extract the upper aqueous layer with an equal volume phenol/chloroform and centrifuge as before. Repeat the extractions until no interface material is seen.
8) Precipitate the aqueous phase by the addition of an equal volume (500ul) of propan-2-ol. Incubate at -20°C for 20 minutes.
9) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes.
10) Wash the RNA once in 70% ethanol and vacuum dry.
11) Re-dissolve in 200ul 0.5% SDS at 65°C.
12) Extract with an equal volume (200ul) of phenol/chloroform as above. Repeat until no interface material is visible.
13) Precipitate pure RNA by the addition of 20ul 3M sodium acetate, 100mM acetate, pH 5.2 and 500ul absolute ethanol. Incubate at -20°C for 20 minutes.
14) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes.
15) Wash the RNA once in 70% ethanol and vacuum dry.
16) Dissolve RNA in appropriate buffer i.e. DEPC-treated, sterile TE, pH 8 or 0.5% SDS if no enzymic manipulation of the RNA is needed. SDS is an inhibitor of ribonucleases.
RNA quality can be assessed by electrophoresis under denaturing conditions using agarose/formaldehyde gels and the MOPS buffer system.
