Obtaining high quality, intact RNA is the first and often the most critical step in performing gene expression analysis. Typically, in order to isolate high quality RNA, the tissue has to be processed immediately after harvest. RNA Solution makes it possible for researchers to postpone RNA isolation for days, weeks, or even months after tissue collection without sacrificing RNA integrity. All we need to do is to add 10 times volume of RNA Solution into the tube containing the freshly collected tissue (1 ml RNA Solution to 100 mg tissue) and store the tube at –20℃ until use. In addition for RNA stabilization, RNA Solution can be easily integrated into a modified single-step RNA isolation method. This modified single-step method isolates undegraded RNA from tissues or cells in hours and can be used to process a large number of samples.
Procedure:
Store 100 mg of tissue or 107 cells (isolated from culture) with 1 ml of RNA Stabilization Solution at –20℃ until RNA isolation.
When processing, thaw and homogenize tissue in RNA Stabilizatio Solution.
Transfer 0.8 ml of the homogenate/cell mix into a 2 ml tube and add 0.8 ml of the acid-phenol, pH 5.2, and 320 �愮 of chloroform.
Vortex the mixture vigorously by mixing 4 times, 30 sec for each.
Centrifuge at 12,000 rpm for 2 min
Transfer the upper aqueous phase (containing RNA) to a fresh 2 ml tube, taking care not to disturb the interface (containing DNA/protein).
Precipitate the RNA by adding an equal volume (0.8 ml) of isopropanol and 80 ml of 3 M NaAc at –20℃ for 30 min
Centrifuge at 12,000 rpm for 15 min and discard the supernatant.
Wash the RNA pellet by using 200 ml of 70% ethanol and gentle Iinverting the tube for several times.
After a brief spin and careful removing the supernatant, let the RNA pellet to air dry for about 5-10 min.
Dissolve the RNA pellet in 20 ml DEPC-treated TE.
Store the samples at –20℃ and used for cDNA synthesis.
