Purification of total RNA from Plant Cells and tissues and filamentous fungi (RNeasy Plant Mini Kit)

Determining the correct amount of starting material

It is essential to use the correct amount of starting material in order to obtain optimal RNA yield and purity. A maximum amount of 100 mg plant material or 1 × 107 cells can generally be processed. For most plant materials, the RNA binding capacity of the RNeasy spin column and lysing capacity of Buffer RLT will not be exceeded by these amounts. Average RNA yield from various plant materials are given in Table 2 (p19). If there is no information about the Nature of your starting material, we recommend starting with no more than 50 mg plant material or 3-4 × 106 cells. Depending on RNA yield and purity, it may be possible to use up to 100 mg plant material or up to 1 × 107 cells in subsequent preparations.

Do not overload the RNeasy spin column, as this will significantly reduce RNA yield and quality

Counting cells or weighing tissue is the most accurate way to quantitate the amount of starting material. As a guide, a 1.5 cm diameter leaf disc weighs 25-75 mg.

Important points before starting

If using the RNeasy Plant Mini Kit for the first time, read “important notes” (p18).

If working with RNA for the first time, read Appendix A (p63).

Fresh or frozen tissues can be used. Tissue can be stored at -70℃ for several months. Flash-freeze tissues in liquid nitrogen, and immediay transfer to -70℃. Do not allow tissues to thaw during weighing or handling prior to disruption in Buffer RLT. Homogenized tissue lysates from step 4 can also be stored at -70℃ for several months. incubate frozen lysates at 37℃ in a water bath until compley thawed and salts are dissolved before continuing with step 5. Avoid prolonged incubation, which may compromise RNA integrity.

The RNeasy Plant Mini Kit provides a choice of lysis buffers: Buffer RLT and Buffer RLC, which contain guanidine thiocyanate and guanidine hydrochloride, respectively. In most cases, Buffer RLT is the lysis buffer of choice due to the greater cell disruption and denaturation properties of guanidine thiocyanate. However, depending on the amount and type of secondary metabolites in some tissues (such as milky endosperm of maize or mycelia of filamentous fungi), guanidine thiocyanate can cause solidification of the sample, making extraction of RNA impossible. In these cases, Buffer RLC should be used.

Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and then place at room temperature (15-25℃).

Buffer RLT, Buffer RLC, and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See page 8 for safety information.

Perform all steps of the procedure at room temperature. During the procedure, work quickly.

Perform all centrifugation steps at 20-25℃ in a standard microcentrifuge. Ensure that the centrifuge does not cool below 20℃.