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
® 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% .
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.
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).
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.
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.
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 or regions: Austria, Benelux, Denmark, Greece, Sweden, Switzerland, China Taiwan, and the United Kingdom.
Marianna Goldrick, Jennifer Ho • Ambion, Inc.