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46
RNeasy Mini Handbook 10/2019
If there is no information about the nature of your starting material, we recommend starting
with no more than 10 mg tissue. Depending on RNA yield and purity, it may be possible to
use up to 30 mg tissue in subsequent preparations.
Do not overload the RNeasy spin column, as this will significantly reduce RNA yield and
quality.
Weighing tissue is the most accurate way to quantitate the amount of starting material. As a
guide, a 3 mm cube (27 mm3) of most animal tissues weighs 30–35 mg.
Important points before starting
If using the RNeasy Kit for the first time, read “Important Notes” (page 19). If working
with RNA for the first time, read Appendix A (page 73).
For optimal results, stabilize harvested tissues immediately in RNAprotect Tissue Reagent
(see protocol on page 41). Tissues can be stored in the reagent for up to 1 day at 37°C,
7 days at 15–25°C, or 4 weeks at 2–8°C, or archived at −30 to −15°C or −90 to
−65°C.
Fresh, frozen, or RNAprotect stabilized tissues can be used. Tissues can be stored at −90
to −65°C for several months. Flash-freeze tissues in liquid nitrogen, and immediately
transfer to −90 to −65°C. 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
−90 to −65°C for several months. Incubate frozen lysates at 37°C in a water bath until
completely thawed and salts are dissolved before continuing with step 5. Avoid
prolonged incubation, which may compromise RNA integrity.
If desired, more than 30 mg tissue can be disrupted and homogenized at the start of the
procedure (increase the volume of Buffer RLT proportionately). Use a portion of the
homogenate corresponding to no more than 30 mg tissue for RNA purification, and store
the rest at −90 to −65°C.
Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and
then place at room temperature.
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