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LeukoLOCK™ Total
RNA Isolation System
Extract MicroRNA from Blood Samples
• Fractionate leukocytes (white
blood cells) from whole blood in a novel closed-tube system
• Eliminate >90% of unwanted globin mRNA by excluding red blood cells
• Stabilize total RNA in leukocytes on filters
• Isolate RNA immediately or after storage at ambient temperature or –20°C
• Standard magnetic bead-based protocol purifies total RNA
• NEW Alternative protocol captures small RNA, including microRNA (miRNA)
Rapidly Fractionate Total White Blood Cells and
Isolate RNA for Expression Profiling
The LeukoLOCK™ Total RNA Isolation System (patent pending) provides a fast way to isolate nucleic acids
from the leukocyte fraction (WBCs) of whole blood. Each leukocyte
filter (originally designed to deplete leukocytes from blood
to prevent graft-versus-host disease in blood transfusion therapy)
can process ~10 ml (range, ~3–30 ml) of whole blood in less than
five minutes. No centrifugation or RBC lysis steps are required;
instead, an empty evacuated blood collection tube is used to
pull the blood through the leukocyte filter in a closed-tube
format. The filter with captured WBCs is then flushed with RNAlater® to
stabilize the RNA, which allows the cells to be safely stored
on the filters at ambient temperature for shipping (several days)
or -20°C for longer time periods (months).
In the standard protocol, flushing the filter
with a guanidinium-based lysis solution disrupts the captured
WBCs, and a simple bead-based procedure is used to isolate RNA
from the resulting lysate. An optional TURBO DNase™ step
can be used to remove trace levels of genomic DNA. This system
is ideal for fractionating WBCs for expression profiling studies
because the time required for fractionation of the blood is minimized,
and the sample is not subjected to effects of centrifugation
or RBC lysis. Also, all the WBC subsets are recovered, in contrast
to density gradient centrifugation, which only recovers PBMCs,
resulting in the loss of mature myeloid cells. Compared to RNA
extraction from whole blood, the LeukoLOCK procedure increases
sensitivity of microarray-based detection because globin mRNAs
(from reticulocytes in whole blood), which can compete with less
abundant mRNA in amplification and hybridization steps of the
analysis, are reduced by more than 90% [1].
NEW PROTOCOL—Isolate RNA that Includes miRNA
An alternative LeukoLOCK protocol has been
developed for efficient purification of small RNAs (<200 nt)
(Figure 1). Instead of magnetic beads, this procedure uses TRI
Reagent® (patented reagent) extraction followed by glass
fiber filter purification to isolate RNA from the WBC lysate.
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Figure 1. Isolate High Quality RNA, Including Small RNA, from Blood with the Alternative LeukoLOCK™ Protocol. Four 9 ml EDTA tubes of blood were drawn from a single donor. Each tube was filtered over a LeukoLOCK filter using the standard closed-tube procedure and then flushed with 2.5 ml of RNAlater®. RNA was extracted from Samples 1 and 2 after 2 hours storage at room temp. Samples 3 and 4 were stored at –20°C for 2 days and then removed from the freezer and stored at room temp for 3 days before RNA extraction. The RNA was extracted using TRI Reagent® followed by purification on glass fiber filters as described in the Alternative LeukoLOCK Protocol. Each sample was eluted in 250 µl. (A) RNA (15 µl or 6% of each prep) was mixed with 8 µl of gel loading solution containing 10 µg/ml ethidium bromide, heated for 5 min at 80°C, and analyzed on a 2% agarose gel. Shorter film exposure, left; longer film exposure, right. (B) Yields and A260/A280 ratios were assessed with a NanoDrop® Spectrophotometer. |
This procedure was used to recover total RNA
including microRNA from blood for use in qRT-PCR experiments
to detect expression of specific
miRNAs in blood using the mirVana™ qRT-PCR miRNA Detection Kit (Figures 2–3). Essentially, the same results were obtained for LeukoLOCK samples
that were stored on the filters at room temperature for 2 hours after fractionation
or samples that were frozen (-20°C) for 2 days followed by 3 days at room
temperature (Figures 1–3).
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Figure
2. Detection of miRNA Using the mirVana™ qRT-PCR
miRNA Detection Kit and Human Blood RNA Purified with
the LeukoLOCK™ Total RNA Isolation Kit. RNA
was extracted from the LeukoLOCK WBC samples using the
alternative protocol for miRNA recovery (as described
in Figure 1) and used as input for detection of several
miRNAs and 5S rRNA with
the mirVana qRT-PCR Detection Kit.
The RNA from each sample was eluted in 250 µl,
and 2 µl was used in a two-step RT-PCR reaction
as directed. Amplicons were detected with SYBR® Green
I (Invitrogen Molecular Probes) on an ABI 7000 thermalcycler.
Each reaction was run in duplicate. Data are displayed
as DCt (Ct-miRNA – Ct-5S rRNA), and this value
was subtracted from 40 so that the height of the bars
shows a positive correlation with miRNA expression levels.
Note, the expression of miR-16 was actually higher than
that of the reference 5S rRNA.
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Figure
3. Representative qRT-PCR Results Using
the mirVana™ qRT-PCR
miRNA Detection Kit and Human Blood RNA Purified with
the LeukoLOCK™ Total RNA Isolation Kit. A
representative amplification plot and dissociation curve
(inset) for the miR-25 amplicon and the primer-dimer
product (no-template control) show specific miRNA amplification.
See legends for Figures 1 and 2 for experimental details.
NTC=No Template Control.
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A video demonstration of the LeukoLOCK filtration
process, as well as a description of the TRI Reagent/silica filter
alternative protocol for recovery of miRNA from blood, is available on our website. [View video]
SuperTaq and SuperTaq Plus are trademarks
of and are manufactured by Enzyme Technologies Ltd. and sold
under licensing arrangements with F. Hoffman-La Roche, Ltd.,
Roche Molecular Systems, Inc. and the Perkin-Elmer Corporation.
Ambion, Inc. is a distributor of Enzyme Technologies, Ltd.,
except in the following countries: Austria, Benelux, Denmark,
Greece, Sweden, Switzerland, Taiwan, and the United Kingdom.
Scientific Contributors
Marianna Goldrick, Jennifer Ho • Ambion,
Inc.
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