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从植物细胞和组织及丝状真菌中提取纯化总RNA

从植物细胞和组织及丝状真菌中提取纯化总RNA

2010/08/17 12:31:40

(用RNeasy Plant Mini Kit)

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 immediately 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 completely 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℃.

Things to do before starting

β-Mercaptoethanol (β-ME) must be added to Buffer RLT or Buffer RLC before use. Add 10 µl β-ME per 1 ml Buffer RLT or Buffer RLC. Dispense in a fume food and wear appropriate protective clothing. Buffer RLT or Buffer RLC containing β-ME can be stored at room temperature for up to 1 month.

Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96-100%) as indicated on the bottle to obtain a working solution.

If performing optional on-column DNase digestion, prepare DNaseⅠstock solution as described in Appendix D (p69).

Produce

  1. Determinate the amount of plant material. Do not use more than 100 mg. 称量植物材料，不要超过100 mg。

Weighing tissue is the most accurate way to determine the amount.

  1. Immediately place the weighed tissue in liquid nitrogen, and grind thoroughly with a mortar and pestle. Decant tissue powder and liquid nitrogen into an RNase-free, liquid-nitrogen-cooled, 2 ml microcentrifuge tube (not supplied). Allow the liquid nitrogen to evaporate, but do not allow the tissue to thaw. Proceed immediately to step 3. 迅速将称量好的材料置于液氮中，充分研磨。将组织细粉与液氮倒入没有RNase并且经液氮冷却的2 ml微量离心管中。可使液氮挥发，但不要将组织解冻。迅速进行步骤3。

RNA in plant tissues is not protected until the tissues are flash-frozen in liquid nitrogen. Frozen tissues should not be allowed to thaw during handling. The relevant procedures should be carried out as quickly as possible.

  1. Add 450 µl Buffer RLT or Buffer RLC to maximum of 100 mg tissue powder. Vortex vigorously. 在最大不超过100的组织细粉中加入450 µl 缓冲液RLT或RLC。剧烈涡旋。

A short 1-3 min incubation at 56℃ may help to disrupt the tissue. However, do not incubate samples with a high starch content at elevated temperatures, otherwise swelling of the sample will occur.

Note: Ensure that β-ME is added to Buffer RLT or Buffer RLC before use.

  1. Transfer the lysate to a QIAshredder spin column (lilac) placed in a 2 ml collection tube, and centrifuge for 2 min at full speed. Carefully transfer the supernatant of the flow-through to a new microcentrifuge tube (not supplied) without disturbing the cell-debris pellet in the collection tube. Use only this supernatant in subsequent steps. 将溶液转移到置于2 ml 收集管中的QIAshredder 旋转柱（淡紫色）中，最大速度离心2 min。小心将液体的上清移至一新的微量离心管中，不要搅动收集管中的细胞残渣沉淀。只用这部分上清进行以下步骤。

It may be necessary to cut off the end of the pipet tip to facilitate pipetting of the lysate into the QIAshredder spin column. Centrifugation through the QIAshredder spin column removes cell debris and simultaneously homogenizes the lysate. While most of the cell debris is retained on the QIAshredder spin column, a very small amount of cell debris will pass through and form a pellet in the collection tube. Be careful not to disturb this pellet when transferring the lysate to the new microcentrifuge tube.

  1. Add 0.5 volume of ethanol (96-100%) to the cleared lysate, and mix immediately by pipetting. Do not centrifuge. Proceed immediately to step 6. 加0.5体积的乙醇（96-100%）至澄清的溶液中，立即用微量移液管上下吹吸混匀。不能离心。很快进行步骤6。

Note: The volume of lysate may be less than 450 µl due to loss during homogenization.

Note: Precipitates may be visible after addition of ethanol. This does not affect the procedure.

  1. Transfer the sample (usually 650 µl), including any precipitate that may have formed, to an RNeasy spin column (pink) placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥8000×g (≥10000 rpm). Discard the flow-through. 将样品（包括形成的沉淀，一般为650 µl）转移到置于2 ml 收集管中的RNeasy旋转柱（粉红）中。轻轻旋紧管盖，在≥8000×g（≥10000 rpm）的速度下离心15 s，丢掉流动的液体部分。

Reuse the collection tube in step 7.

If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation. Flow-through contains Buffer RLT, Buffer RLC, or Buffer RW1 and is therefore not compatible with bleach. See page 8 for safety information.

Optional: If performing optional on-column DNase digestion (p23), follow steps D1-D4 (p69) after performing this step.

  1. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000×g (≥10000 rpm) to wash the spin column membrane. Discard the flow through. 加700 µl缓冲液RW1到RNeasy旋转柱中。轻轻旋紧管盖，在≥8000×g（≥10000 rpm）的速度下离心15 s，对旋转柱膜进行清洗。丢掉流动的液体部分。

Reuse the collection tube in step 8.

Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely.

  1. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000×g (≥10000 rpm) to wash the spin column membrane. Discard the flow through. 加700 µl缓冲液RPE到RNeasy旋转柱中。轻轻旋紧管盖，在≥8000×g（≥10000 rpm）的速度下离心15 s，对旋转柱膜进行清洗。丢掉流动的液体部分。

Reuse the collection tube in step 9.

Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use.

  1. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000×g (≥10000 rpm) to wash the spin column membrane. 加700 µl缓冲液RPE到RNeasy旋转柱中。轻轻旋紧管盖，在≥8000×g（≥10000 rpm）的速度下离心2 min，对旋转柱膜进行清洗。

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 form the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will over.

  1. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. 可选步骤：将RNeasy旋转柱置于新的2 ml 收集管中，把旧的收集管连同其中的液体一同丢掉。轻轻旋紧管盖，在最大速度下离心1 min。

Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 9.

  1. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30-50 µl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥8000×g (≥10000 rpm) to elute the RNA. 将RNeasy旋转柱置于新的1.5 ml收集管中。在旋转柱中直接加30-50 µl无RNase的水。轻轻旋紧管盖，在≥8000×g（≥10000 rpm）的速度下离心1 min，对RNA进行稀释。

  2. If the expected RNA yield is ＞30 µg, repeat step 11 using another 30-50 µl RNase free water, or using the eluate from step 11 (if high RNA concentration is required). Reuse the collection tube from step 11. 如果预期RNA产量＞30 µg，用30-50 µl无RNase水重复步骤11，或者用第11步产生的稀释液重复步骤11（如果需要高浓度的RNA）。重复使用步骤11的收集管。

If using the eluate from step 11, the RNA yield will be 15-30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.