Bio-Rad Experion Protein Analysis Kits - Manual (Page 22)

Starter Kit Test 2 — Quantitation Across a Range of
Concentrations
2.1 Overview of Test 2
In this test, you analyze a dilution series of the total RNA sample to examine the
reproducibility of quantitation across the entire quantitation range of the Experion
™ RNA
StdSens analysis kit (25–500 ng/µl).
Note: The quantitative range of the Experion RNA StdSens analysis kit is different from its
detection range: whereas the detection range of the assay for total RNA is 5–500 ng/µl, the
quantitative range is 25–500 ng/µl (at the lower end of the quantitative range, the
signal-to-noise ratio is lower). Though RNA detection and purity analysis can be performed
at concentrations of less than 25 ng/µl using the StdSens assay, quantitation at these low
concentrations is not recommended since RNA concentrations of <25 ng/µl are outside the
linear range of detection. For quantitation of samples containing 0.1–5 ng/µl RNA, use the
Experion RNA HighSens analysis kit.
2.2 Assay Procedure
1. Set up the electrophoresis station, clean the electrodes, thaw and equilibrate the
reagents, and prepare the gel (G) and gel-stain solution (GS) as outlined in Sections
1.2.1–1.2.3. If the G has already been prepared, equilibrate it as detailed in Section
1.2.2.
2. Denature the total RNA sample and RNA ladder as described in Section 1.2.4, steps
1–2, using 3 µl RNA ladder and 7 µl total RNA sample (stock preparation). Label the
total RNA sample “S1”.
3. This test uses a dilution series of the total RNA sample. Label 3 microcentrifuge tubes
(S1–S3) and prepare the following dilutions:
Tube S1 (500 ng/µl): No additions; this is the stock preparation you denatured
Tube S2 (100 ng/µl): 8 µl DEPC-treated water + 2 µl total RNA from tube S1
Tube S3 (25 ng/µl): 6 µl DEPC-treated water + 2 µl total RNA from tube S2
4. Vortex to mix the tubes, and spin down.
5. Prime and load the chip as described in Sections 1.2.5 and 1.2.6 and using the chip
layout shown in Figure 2.1.
S3 S2 S1 G
S3 S2 S1 GS
S3 S2 S1 GS
S3 S2 S1 L
Fig. 2.1. Chip layout for Experion RNA StdSens starter kit Test 2. S1–S3, sample numbers; GS, gel-
stain solution; G, RNA gel; L, RNA ladder.
6. Run the RNA StdSens analysis as described in Section 1.2.7. Enter the sample names
and information appropriate to the chip layout in Figure 2.1.
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