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.
b. Centrifuge for ~30 sec to pass the solution through the filter.
c. Repeat with a second 180 μl aliquot of Wash Solution 2/3.
8. Discard the flow-through and centrifuge the filter for 1 min
a. Open the Micro Filter Cartridge assembly, remove the filter cartridge from the Collection Tube, and pour out the flow-through.
b. Replace the Micro Filter Cartridge into the same Collection Tube, close the cap, and centrifuge the assembly for 1 min to remove residual fluid and dry the filter.
9. Elute the RNA in 2 x 8–10 μl of Elution Solution
a. Label a Micro Elution Tube (1.5 ml tubes provided with the kit) and transfer the Micro Filter Cartridge into it.
b. Apply 8–10 μl of Elution Solution, preheated to 95°C, to the center of the filter. Since accurate pipetting of hot liquids can be difficult, pipet the Elution Solution up an down every time a new tip is used.
c. Close the cap and store the assembly for 5 min at room temp.
d. Centrifuge the assembly for 1 min to elute the RNA from the filter. Tension from the hinge of the Micro Elution Tube can occasionally cause the cap to pop off during the elution spin. To minimize the chance of this happening, bend the cap hinge back and forth several times, then press the cap securely onto the Micro Filter Cartridge. e. Repeat steps b–d with a second 8–10 μl of preheated Elution Solution and collect the eluate in the same Micro Elution Tube.
NOTE
The exact volume of Elution Solution used is not critical and may be adjusted according to user preference. In general, 85–90% of the RNA bound to the filter will be recovered using 2 x 8–10 μl of Elution Solution.
(optional) DNase I Treatment and DNase Inactivation
NOTE
This DNase treatment is optional but recommended if the RNA will be used in RT-PCR assays or other applications where it is desirable to remove trace amounts of contaminating genomic DNA.
1. Add 1/10 volume of 10X DNase I Buffer and 1 μl of DNase I
Add 1/10th volume 10X DNase I Buffer (e.g. 2 μl for RNA eluted in 20 μl) and 1 μl of DNase I (provided with the kit) to the sample and mix gently but thoroughly.
2. Incubate 20 min at 37°C • Incubate the DNase reaction for 20 min at 37°C. A 30 min incubation may be needed for treating RNA derived from >100,000 cells or from tissue samples larger than ~3 mg.
• Remove the DNase Inactivation Reagent from –20°C and allow it to thaw at room temp during this incubation.
3. Add 2 μl or 1/10 volume DNase Inactivation Reagent, mix well, and leave at room temp for 2 min
a. Vortex the DNase Inactivation Reagent vigorously to completely resuspend the slurry.
b. Use 2 μl or 1/10 volume DNase Inactivation Reagent, whichever is greater. For example, if the RNA volume is 50 μl, and 1 μl DNase I and 5 μl of DNase Buffer were used in step 1, add 5.6 μl of DNase Inactivation Reagent.
NOTE
The DNase Inactivation Resin may become difficult to pipette after multiple uses due to depletion of fluid from the interstitial spaces. If this happens, add a volume of Elution Solution equal to approximately 10–20% of the remaining DNase Inactivation Reagent and vortex well to recreate a pipettable slurry.
c. Store the reaction at room temp for 2 min, vortexing once during this interval to disperse the DNase Inactivation Reagent.
4. Pellet the DNase Inactivation Reagent and transfer the RNA to a fresh tube
Centrifuge the reaction 1.5 min at maximum speed to pellet the DNase Inactivation Reagent. Transfer the RNA to a fresh RNase-free tube (not supplied with the kit) and store at –20°C.
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