Bio-Rad Experion Protein Analysis Kits - Manual (Page 11)

– RNA loading buffer (yellow cap)
– RNA gel (green cap) 2
Notes: Experion RNA stain (blue cap) is light-sensitive and contains DMSO, which is highly
hygroscopic. Cap the tube of stain tightly and keep it covered (for example, with foil) at all
times to avoid exposure to light.
Inspect the reagents prior to thawing: if the RNA gel (green cap) has been frozen, do not
use it.
Do not store reagents at room temperature for more than 2 hr, as this will affect their shelf
life.
3. Vortex the contents of each tube and briefly centrifuge to collect the solutions to the
bottoms of the tubes. Make sure that the RNA stain solution (blue cap) is completely
thawed before proceeding.
4. Prepare 3 µl aliquots of the RNA ladder in RNase-free microcentrifuge tubes. Freeze
all aliquots except the one you will be using at –80°C. This ensures integrity of the
RNA ladder, a critical component of the RNA StdSens assay.
1.2.3 Filter the Gel and Prepare the Gel-Stain Solution
Notes: The Experion RNA StdSens starter kit includes 1 spin filter, which is sufficient for
one preparation of filtered gel (G). You will also use the prepared G in Test 2. The prepared
G can be used for up to 4 weeks when stored in the dark at 4°C between each use. The
gel can be refiltered once after that to extend its use for another 4 weeks.
Additional spin filter tubes and reagents may also be purchased separately (see Appendix
D for ordering information).
This protocol generates enough GS for 3 chips. Increase the amount, if required, by using
a 65:1 ratio of gel and stain. Prepare fresh GS daily.
1. Filter the gel. Pipet 600 µl RNA gel (green cap) into a spin filter and label the tube with
“G” and the date. Centrifuge at 1,500 x g for 10 min. Confirm that all of the gel has
passed through the filter and then discard the filter.
2. Prepare the GS. Pipet 65 µl filtered gel into an RNase-free 0.65 ml microcentrifuge
tube and label the tube “GS”. Add 1 µl RNA stain and vortex. Keep the GS protected
from light.
1.2.4 Prepare the Samples and RNA Ladder
1. Once the total RNA sample and RNA ladder have thawed, vortex the tubes briefly and
spin down for a few sec. The concentration of the stock total RNA preparation is
500 ng/µl.
2. Denature the RNA sample and the RNA ladder. Transfer 4 µl total RNA sample and
3 µl RNA ladder to separate 0.65 ml RNase-free microcentrifuge tubes labeled “S1”
and “L”, respectively. Heat the tubes at 70°C for 2 min, and place them on ice
immediately after heating. Vortex briefly and spin down. Keep the tubes on ice.
3. Generate the 100 ng/µl sample used in this test by adding 16 µl DEPC-treated water
to the sample tube (S1).
4. Vortex to mix the tubes, and spin down.
2 If the filtered gel (G) was prepared previously, remove it from storage and equilibrate it to room temperature.
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