If the expected RNA yield is >30 μg, repeat Step 9 using another 30–50 μL of RNase-free water.
Alternatively, use the eluate from Step 9 (if high RNA concentration is required). Reuse the collection tube from Step 12.
Disruption using the TissueLyser II, TissueLyser LT, or TissueRuptor.
For detailed information on disruption of plant tissues for purification of RNA, see TissueLyser Handbook, TissueLyser LT Handbook, or TissueRuptor Handbook. (The RNeasy Mini Handbook will be updated with this option.)
Transfer up to 700 µL of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 mL collection tube (supplied).
Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through. If the sample volume exceeds 700 µL, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.
See “Disrupting and homogenizing starting material”, pages 18–21, for more details on disruption and homogenization.
Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to do before starting”).
After storage in RNAlater RNA Stabilization Reagent, tissues may become slightly harder than fresh or thawed tissues. Disruption and homogenization using standard methods is usually not a problem. For easier disruption and homogenization, we recommend using 600 µl Buffer RLT.
Note: Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column. Homogenization with the TissueLyser LT, TissueLyser II, and rotor–stator homogenizers generally results in higher RNA yields than with other methods.
Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane.
The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.
Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
Carefully remove the supernatant by pipetting, and transfer it to a new microcentrifuge tube (not supplied).
Use only this supernatant (lysate) in subsequent steps. In some preparations, very small amounts of insoluble material will be present after the 3 min centrifugation, making the pellet invisible.
RNA in harvested tissues is not protected until the tissues are treated with RNAlater RNA Stabilization Reagent, flash-frozen, or disrupted and homogenized in step 3. Frozen tissues should not be allowed to thaw during handling. The relevant procedures should be carried out as quickly as possible.
Note: Remaining fresh tissues can be placed into RNAlater RNA Stabilization Reagent to stabilize RNA (see protocol on page 34). However, previously frozen tissues thaw too slowly in the reagent, preventing the reagent from diffusing into the tissues quickly enough to prevent RNA degradation.
Remove RNAlater stabilized tissues from the reagent using forceps. Determine the amount of tissue. Do not use more than 30 mg.
Weighing tissue is the most accurate way to determine the amount.
Note: If the tissues were stored in RNAlater Reagent at –20°C, be sure to remove any crystals that may have formed.
The Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT Fast System) uses fluorescent-based PCR chemistries to provide:
You can perform several assay types on the 7900HT Fast System using reactions plates in the 96-well, 384-well, or TaqMan® Low Density Array format. This guide describes the allelic discrimination assay.
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