Laboratory of Christopher E. Mason, PhD Prepared by Paul Zumbo, 1/9/11 Purify Total RNA (< 45 ug) with DNase Treatment This process purifies and concentrates RNA (< 45 ug) and removes contaminant DNA using silica-­‐
based membrane technology and DNase digestion. All RNA molecules < 200 nt are selectively excluded. Add DNase I
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Microcentrifuge (with rotor for 2 ml tubes) Vortex mixer Consumables §
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RNase-­‐free water Disposable gloves RNase-­‐free Ethanol Sterile, RNase-­‐free pipet tips DNase kit (Qiagen, 79254) MinElute columns (Qiagen, 74004) RNase-­‐free tubes Preparation §
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Add Buffer RLT
and ethanol
Bind RNA
RNA
Wash
80% ethanol DNase I stock solution Buffe RPE Elute
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Adjust sample volume to 87.5 ul with RNase-­‐free water. Add 10 ul Buffer RDD to 2.5 ul DNase I stock solution. Mix by gentle inversion, and briefly spin. Transfer 12.5 ul of DNase mix to sample. Mix well by gentle inversion, and briefly spin. Incubate at RT (15-­‐25 ºC) for 15 min. Add 350 ul Buffer RLT, vortex, and briefly spin. Add 250 ul 100% EtOH, vortex, and briefly spin*. Transfer sample (700 ul) to an RNeasy MinElute spin column placed in a 2 ml collection tube. Let sit for 2 min at RT (15-­‐25 ºC). Close lid, and centrifuge at 10,000 rpm for 30 s at RT (15-­‐
25 ºC). Discard flow-­‐through. Add 500 ul Buffer RPE to spin column. Close lid, and centrifuge at 10,000 rpm for 30 s at RT (15-­‐25 ºC). Discard flow-­‐through. Add 500 ul 80% EtOH to spin column. Close lid, and centrifuge at 10,000 rpm for 2 min at RT (15-­‐25 ºC). Discard flow-­‐through and collection tube. Place spin column in a new 2 ml collection tube. Open the lid of the spin column (orient so that they point in the direction opposite the rotation of the rotor; leave at least one empty position between columns), and centrifuge at 13,200 rpm for 5 min at RT (15-­‐25 ºC). Place spin column in a new 1.5 ml collection tube. Let sit for 5 min at RT (15-­‐25 ºC) with lid open to completely evaporate residual ethanol. Add 19 ul RNase-­‐free water directly to the center of the spin column membrane. Let sit at RT (15-­‐25 ºC) for 2 min, and centrifuge at 13,200 rpm for 1 min at RT (15-­‐25 ºC). Pipet flow-­‐through from the collection tube (17 ul) back onto the spin column membrane. Let sit at RT (15-­‐25 ºC) for 2 min, and centrifuge at 13,200 rpm for 1 min at RT (15-­‐25 ºC). * Avoid prolonged centrifugation, which will drive the RNA through the ethanol solution to the wall of the tube. 1