Thermo Scientific KingFisher Pure RNA Blood Kit, Инструкция по эксплуатации

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24 | Thermo Scientific KingFisher Pure RNA Blood Kit Thermo Fisher Scientific
Chapter 5 | Protocols and Pipetting Instructions
Instructions for Total RNA Purification
from Tempus Stabilized Blood Samples
1. Collect and store the blood samples in Tempus tubes according to the
manufacturer’s instructions.
2. If the sample is frozen, thaw the sample in the Tempus tube at room
temperature according to the manufacturer’s instructions.
3. Transfer the entire contents of the Tempus tube into a 50 ml conical
tube. Bring the total volume to 12 ml by adding the required amount of
PBS. Typically a Tempus stabilized blood sample volume is 9 ml, which
requires the addition of 3 ml of PBS to bring the total volume to 12 ml.
4. Vortex efficiently for 30 s to ensure proper mixing of the contents.
5. Centrifuge the appropriate volume (1−2 ml) of Tempus blood lysate in
conical tubes at maximal speed (about 12,000−16,000 × g) for 10 min
at 4°C. If the Tempus blood volume is more than 2 ml, increase the time
of centrifugation up to 30 min.
6. Carefully discard the supernatant from each tube and add 450 µl of
Lysis Buffer (supplemented with DTT) and 20 µl of Proteinase K. Vortex
the samples and incubate at room temperature for 10 min. Continue
the purification as in
“Instructions for KingFisher Flex with 96 Deep Well
Plates” on page 18 or as in “Instructions for KingFisher Duo with
12-pin Magnet Head” on page 20 .
Quantification and Determination of
the Purity of RNA
It is recommended to measure the absorbance at 320 nm, 280 nm, and
260 nm. The concentration of RNA can be defined with the absorbance at
260 nm (A
260
). One unit at 260 nm corresponds to 40 μg of RNA per ml. The
ratio between the A
260
/A
280
indicates the purity of the RNA. The value for RNA
should be ≥ 1.8–2.1.