RNA Isolation Protocol from LCM Samples

1. ｄｒｏｐ sample into 100 μl Lysis Solution and incubate for 30 min at 42°C

Using sharp forceps, peel off the thermoplastic film containing the captured cells and ｄｒｏｐ it into a 0.5 ml microfuge tube containing 100 μl of Lysis Solution. Make sure that the sample is completely immersed in

Lysis Solution. a. Incubate the sample for 30 min at 42°C. b. Vortex briefly to mix. Briefly centrifuge to collect the fluid at the bottom of the tube.

2. Prewet the Filter with 30 μl of Lysis Solution for >= 5 min

a. Prewet the Micro Filter Cartridge Assembly by applying 30 μl of Lysis Solution to the center of the filter and allow it to soak while performing the next two steps. b. After completing steps 3 and 4 below, or after 5 min, centrifuge the prewetted filter for ~30 seconds at top speed to remove liquid.

3. Add 3 μl of LCM Additive to the lysate and mix

a. Add 3 μl of the LCM Additive to the lysate and mix well by briefly vortexing.

b. Briefly centrifuge to collect the fluid at the bottom of the tube.

4. Add 100% ethanol to recover either only large RNA species or all RNA

The amount of ethanol added to the lysate mixture determines what size of RNA will be captured on the filter. Instructions are provided below for recovering only RNA species larger than ~75 bases, or for recovering both large and small RNA species, including tRNAs and microRNAs.

• To recover only large RNA species:

Add 0.5 volumes (52 μl) of 100% ethanol to the lysate mixture, and mix by pipetting up and down or by gently vortexing.

• To recover

large and small RNA species:

Add 1.25 volumes (129 μl) of 100% ethanol to the lysate mixture, and mix by pipetting up and down or by gently vortexing. (Yields of total RNA using this method may vary from those obtained using

the above method.)

5. Pass the lysate mixture through a prepared Micro Filter Cartridge Assembly

Be sure to remove the Lysis Solution used to prewet the filter by centrifugation (step 2.b) before applying the lysate mixture. a. Load the entire lysate/ethanol mixture onto the prepared Micro Filter Cartridge Assembly and close the cap. b. Centrifuge for 1 min at 10,000 x g to bind the RNA to the filter.

6. Wash with180 μl of Wash Solution 1

a. Add 180 μl of Wash Solution 1 (working solution mixed with ethanol) to the filter and close the cap.

b. Centrifuge for 1 min at 10,000x g to pass the solution through the filter.

NOTE

All centrifugation in the following steps should be done at 13,000 X g, or

maximum speed in a microcentrifuge.

7. Wash the filter with 2 x 180 μl Wash Solution 2/3

a. Open the Micro Filter Cartridge, add 180 μl of Wash Solution 2/3 (working solution mixed with ethanol) to the filter and close the cap.

NOTE

Sometimes a precipitate will form in Wash Solution 2/3 over time. Avoid

removing the crystals of precipitated material at the bottom of the bottle

when removing the Wash Solution for use. There is no need to redissolve

the precipitate.