RNeasy kit

Wash the column with 700 pL of buffer RWl (from RNeasy kit) and centrifuge sample for 60 s at >8000 x g. Discard the flow-through. 

Transfer RNeasy column into a new 2-mL collection tube. Apply 500 pL of RPE buffer (from RNeasy kit). Wash the coluum by centrifugation at >8000 x g for 60 s. Discard flow-through. 

Add the chosen disruption buffer (RLT, supplied with RNeasy kit, or Trizol) to the tissue or cells in a clean tube. Use the needle tip to begin the disruption of the tissue, then carefully draw tissue suspension up and down the syringe at least five times or until it appears smooth. Apply this suspension to a QIAshredder column placed in a 2-mL collection tube, and centrifuge at maximum speed for 2 min or until all homogenate has passed through the Qiashredder column. 

Follow the instructions in the Qiagen RNA RNeasy Kit to purify fragmented RNA, and elute in 50 pi DEPC treated H2O. 

Purify products by using a RNA isolation column (e.g., Qiagen RNeasy Kit, see Subheading 3.2), and elute in 40 pi DEPC treated H2O (re Note 3). 

Several kits are commercially available for the extraction of RNA from plant, bacteria, or mammalian sources. We have used the RNeasy Mini Kit (Qiagen, Valencia, CA) for the extraction of RNA from bacteria following the procedure recommended by the manufacturer (92). This kit relies on guanidine thiocyanate-silica isolation of RNA. Here, guanidine thiocyanate serves as a chaotropic agent, which both lyses cells and inactivates nucleases. In its presence, the released nucleic acids bind to silica particles, which provide a solid phase from which the collected RNA can later be eluted using water (93). This kit is suitable for the isolation and purification of up to 100 pg RNA molecules >200... 

Because microarray analysis requires the use of fluorescence, the lysis buffer must be chosen carefully. For instance, many lysis reagents contain phenol and chloroform. Residual phenol and or chloroform can interfere with fluorescent labeling. This usually equates to less signal and more background and hence difficulty in interpreting microarray results. The lysis buffer RLT (provided with the RNeasy Mini Kit) does not interfere with subsequent labeling of the samples. 

RNeasy Plant Mini Kit (Qiagen)—this kit contains columns and the RLT and RPE solutions used in purifying RNA. 

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