It is essential to produce highly purified RNA free of proteins, polysaccha-ride, cell debris, and particularly RNAses. The former contaminants can interfere with the efficiency of synthesis of biotinylated cRNA and also can bind nonspecifically to probes, resulting in increased background and the masking of low frequency target signals.
1. TRIzol Reagent (Invitrogen; cat. no. 15596) Store at 4°C, stable for 6 mo. Safety information: toxic in contact with skin and if swallowed. TRIzol causes burns (see Note 1).
2. Chloroform (BD; cat. no. 10077). Store at RT, stable for minimum of 5 yr. Safety information: harmful by inhalation and if swallowed. Irritating to skin. Possible risk of irreversible effects exists. Danger of serious damage to health by prolonged exposure.
4. 100% Ethanol (BD; cat. no. 10107). Store at RT, stable for minimum of 5 yr. Safety information: highly flammable. Keep away from sources of ignition. Keep container tightly closed.
5. 75% Ethanol (make up using 100% ethanol and DEPC H2O).
6. 1.5-mL Microfuge tubes.
7. Heating block or waterbath suitable for 1.5-mL Microfuge tubes.
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