| What the reagent is | A monophasic solution containing phenol and guanidinium thiocyanate. It disrupts cells and helps denature proteins, including RNases that can degrade RNA. | Cellular material is lysed and nucleic acids and proteins are released into the homogenate. | Choose a formulation intended for the sample type and downstream application; check its instructions for compatible input amounts and handling requirements. |
| Sample compatibility | Commonly used for cultured cells, animal tissues, and other biological samples that can be thoroughly homogenized. | Successful extraction depends on complete disruption and a sample-to-reagent amount within the protocol’s stated range. | For fibrous, fatty, or high-polysaccharide samples, consider whether the protocol includes suitable homogenization and cleanup steps. Avoid overloading the reagent. |
| Initial homogenization | The sample is disrupted in the reagent to produce a uniform lysate and expose cellular components to the denaturing solution. | A homogeneous lysate reduces the risk of incomplete extraction and uneven phase separation. | Use clean, RNase-controlled equipment and follow the protocol’s recommended disruption method. Keep samples cold when specified by the protocol. |
| Phase-separation trigger | Chloroform is added to the homogenate, mixed, and then centrifuged. This creates distinct liquid phases. | Many standard protocols use approximately 0.2 mL chloroform per 1 mL of reagent, but the required ratio is protocol-dependent. | Use the exact reagent-to-chloroform ratio, mixing method, incubation time, temperature, and centrifugation conditions specified for the selected procedure. |
| Upper aqueous phase | Under acidic phenol–guanidinium conditions, most RNA partitions into the upper aqueous phase. | This phase is the fraction normally collected for total RNA purification. | Transfer only the aqueous layer. Avoid disturbing the interface, because carryover can introduce DNA, proteins, or organic-phase contaminants. |
| Interphase | The interface between the aqueous and organic phases contains most of the precipitated genomic DNA. | DNA may be recovered from this fraction using an appropriate protocol. | Do not aspirate the interphase when the goal is clean RNA. DNA-sensitive downstream assays may require additional DNase treatment, as appropriate. |
| Lower organic phase | The lower phase contains phenol and other organic components, with most proteins and lipids partitioning into it or the interphase. | This fraction is not the RNA-containing fraction in the standard total-RNA workflow. | Keep it separate from the aqueous layer. Handle and dispose of it as hazardous chemical waste according to local rules. |
| RNA precipitation and wash | RNA in the aqueous phase is commonly precipitated with isopropanol, collected by centrifugation, and washed with dilute ethanol. | A washed RNA pellet is obtained for resuspension; precipitation and wash volumes vary by protocol. | Follow the specified precipitation time and centrifugation conditions. Remove residual ethanol before dissolving the pellet, without overdrying it. |
| RNA quality checks | Purity and integrity are assessed after extraction using suitable spectrophotometric and/or electrophoretic methods. | Absorbance ratios can indicate contamination, while electrophoresis or an RNA integrity instrument evaluates degradation. | Interpret measurements together with the downstream assay requirements; absorbance ratios alone do not establish RNA integrity or suitability. |
| Safety and storage | Phenol and chloroform are hazardous chemicals; guanidinium-containing waste must not be mixed with bleach. | Safe handling requires appropriate chemical controls and separate waste streams. | Use a chemical fume hood, suitable personal protective equipment, and the current safety data sheet. Store the reagent as directed by its manufacturer. |