RNA isolation procedure

Day 1

Starting Material:
~30 mg of animal tissue (25-35 mg)

Reagents and disposable Materials:
1.5 ml microcentrifuge tubes
2 ml microcentrifuge collection tubes
Micropipette tips
RNA isolation mini-column
Weighing Paper
Disposable Scalpel
Ice
RNA extraction lysis buffer (with 1% beta-mercaptoethanol)
RNA isolation mini-column wash solution
RNA isolation mini-column rinse solution
RNase-free distilled water
70% ethanol (RNase-free)

RNA isolation procedure

2) Using clean new disposable scalpel, weighing paper and scale, and keeping the tissue and grinder in ice, quickly isolate  ~30 mg of animal tissue (25-35 mg) and add to tissue grinder and lysis buffer.

3) While keeping on ice, homogenize the tissue by vertical plunging and twist-grinding until a uniform homogenate arises (20 to 40 seconds).

4) Transfer to 1.5 ml microcentrifuge tubes and centrifuge at room temperature for three minutes @ 12K to pellet the cellular debris.

5) While step 4 is in progress, add 600 ul of room temperature 70% ethanol (RNase-free) to a 1.5 ml tube.

6) When the centrifugation is complete, carefully add ~ 600 ul of the raw extract (supernatant) to the 70% ethanol and mix by three to five slow rounds of micropipetting.

7) Add immediately to the RNA isolation mini-column and centrifuge at room temperature for 15 seconds @ 10K to load the RNA onto the column.  Discard the flow-through liquid (in fumehood).  Add the remainder of the raw extract and repeat the room temperature centrifugation for 15 seconds @ 10K.

8) Add 700 ul of wash solution and centrifuge at room temperature for 15 seconds @ 10K to wash the column.  Discard flow-through liquid and collection tube.

9) Place loaded RNA isolation mini-column in fresh 2 ml microcentrifuge collection tube and add 500 ul of the RNA isolation mini-column rinse solution and centrifuge at room temperature for 15 seconds @ 10K to rinse the column.  Discard flow-through liquid. Repeat the addition of 500 ul of the RNA isolation mini-column rinse solution and centrifuge at room temperature for 2 minutes @ 10K to dry the column. Discard flow-through liquid and recentrifuge the column to ensure that the column is dry.

10) Transfer column to a 1.5 ml microcentrifuge tube.  Add 30 ul of RNase-free distilled water and centrifuge at room temperature for one minute @ 10K to collect the total RNA.

11) Place on ice for immediate use or store at -70 C for later use.

12) Take one ul of the sample (added to 99 ul of RNase-free distilled water) to measure concentration.