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134 Mokany et al.
3.2. Calibration Curves for BCR
Calibration curves for quantifying BCR transcripts are constructed by diluting
Meg-01 total RNA in Nuclease-free water.
1. Make up multiple aliquots of 120 µL of 100 ng/µL Meg-01 total RNA and store
frozen as Calibrator 1 for BCR curve. Calibrators 2–5 are made by performing
fourfold serial dilutions of Calibrator 1 as follows:
a. For Calibrator 2 (25,000 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 1
to 15 µL of nuclease-free water.
b. For Calibrator 3 (6250 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 2 to
15 µL of nuclease-free water.
c. For Calibrator 4 (1563 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 3 to
15 µL of nuclease-free water.
d. For Calibrator 5 (391 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 4 to
15 µL of nuclease-free water.
3.3. Extraction and Quantitation of Total RNA
All RNA extractions are performed in a biohazard-protection hood (see Note
7). Two different methods are described to extract total RNA: (1) from patient
samples, and (2) from cultured cells.
3.3.1. Extraction of RNA From Patient Specimens
1. For the extraction of RNA from patient specimens, use TRIzol reagent (Life
Technologies) according to the manufacturer’s recommendations. Be sure to
work quickly and keep patient samples ice-cold to minimize degradation.
2. To measure concentration of total RNA, centrifuge at 10,000g for 15 s and return
to ice. Measure the absorbance of a sample of the concentrated stock at 260 nm on
the ultraviolet spectrophotometer (following manufacturer’s instructions for use).
Calculate the concentration of the RNA solution using the following formula:
Concentration (µg/mL) = 40 × A 260 × dilution factor
3. Where possible, dilute diagnostic RNA samples (pretreatment) to 10 ng/µL and
follow-up samples (posttreatment) to 100 ng/µL in nuclease-free water. Aliquot
at least two 12-µL single-use samples (allows 5-µL duplicates to be analyzed per
experiment and repeated) to avoid freeze thawing of samples (see Note 8).
4. Keep concentrated stocks of total RNA on dry ice at all times and return to –80°C
storage when completed. Smaller aliquots of the concentrated stock can be made
where possible to avoid freeze thawing of the entire sample for further analysis
of other transcripts of interest.
3.3.2. Extraction of Total RNA From Cultured Cells
For extraction of total RNA from cultured cells, follow the instructions of
“QIAamp RNA mini protocol” (“QIAamp RNA blood mini handbook” from