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Letters |
1
Department of Pathology, University of California, Irvine, CA 92717,
2
Section of Thoracic Surgery, School of Medicine, Keio University, Tokyo 108, Japan,
3
Department of Psychiatry, University of California, Irvine, CA 92717,
4
Hitachi Chemical Research Center, Irvine, CA 92612
a Address correspondence to this author at: Hitachi Chemical Research Center, 1003 Health Sciences Road West, Irvine, CA 92612. Fax 714-725-2727; e-mail mmitsuha{at}uci.edu.
To the Editor:
The purification of intact RNA is the first critical step for mRNA analysis. Because of the instability of RNA, the process requires labor-intensive multiple steps while guarding against RNases. Although many techniques and commercial kits are available, these are techniques for research laboratories and are not suitable when RNA is collected from patients in clinics, hospitals, operating rooms, or even the emergency room and stored until use.
Here we report a rapid method for the preparation of RNA from
blood. Mononuclear leukocytes were first separated by conventional
density gradient centrifugation or equivalent methods. The cell
suspensions were filtered through glass fiber membranes, and the
resultant membranes were immediately dried under vacuum. After dried
membranes were stored at room temperature for 1 week, RNA was isolated
by acid-guanidine/phenol/chloroform (AGPC) extraction (1).
As a control, cell suspensions were centrifuged, and pellets were
homogenized by AGPC. The quality of RNA was tested by three criteria:
(a) agarose gel electrophoresis to confirm 18S and 28S rRNA
bands (2), (b) measurement of total mRNA by the
method we developed (3), and (c) reverse
transcription-PCR (RT-PCR) (4) of glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) mRNA. As a result, rRNAs and G3PDH-specific mRNA
in dried samples were stable at room temperature for 1 week (Fig. 1
A), and the amount of total mRNA was similar to that of the
control (mean ± SE, 5.3 ± 0.4 vs 4.6 ± 0.6 µg/L
blood). More importantly, RNA was easily released from membrane-trapped
cells by simply passing them through AGPC solution, whereas vigorous
homogenization (pipetting/vortex-mixing) was required to recover RNA
from cell pellets in control experiments.
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In contrast to mononuclear leukocytes, whole blood is much more difficult to handle, probably because of the presence of RNase-rich granulocytes. The stability of RNA may be improved by selective lysis of erythrocytes before recovery of the nucleated cells by centrifugation (5). In the present study, erythrocytes were selectively lysed in hypotonic solution followed by filtration through glass fiber filter membranes. When cell-trapped membranes were dried and were stored at room temperature, we identified substantial amounts of degradation of RNA, although some rRNA bands remained (data not shown). We also tested various organic solvents to accelerate drying in conjunction with RNase inhibitors, but none of the procedures prevented RNA degradation (data not shown). We then applied a denaturing solution [4 mol/L guanidine thiocyanate, 25 mmol/L sodium citrate, 5 g/L N-lauroylsarcosine (Na salt, Sarkosyl), and 0.1 mol/L 2-mercaptoethanol] to the membranes immediately after cells were trapped, and the RNA-containing pass-through fraction (guanidinium-stabilized RNA) was stored at room temperature for 2 weeks. RNA was then isolated by phenol/chloroform extraction.
As shown in Fig. 1B
, we obtained high-quality RNA even after a 2-week
storage period at room temperature in terms of rRNA bands in agarose
gel electrophoresis (Fig. 1B
, upper panel) and RT-PCR of G3PDH (Fig. 1B
, lower panel). Furthermore, there were no differences in the amounts
of total mRNA among 0, 1, 7, and 14 days after ambient storage
(mean ± SE, 16.2 ± 3.9, 15.7 ± 1.5, 13.8 ± 3.2,
and 14.5 ± 3.0 µg/L blood, respectively). In control
experiments, leukocytes were pelleted by centrifugation, followed by
conventional RNA extraction (1). However, we could recover
neither rRNA (Fig. 1B
, upper panel) nor mRNA from whole blood (<1
µg/L blood), and subsequent RT-PCR failed to amplify G3PDH mRNA (Fig. 1B
, lower panel).
By capturing cells with glass fiber membranes, we could eliminate centrifugation, which is a major hurdle when assay automation is considered. Furthermore, this method overcomes the problems of irreproducibility of homogenization of cell pellets. In the case of mononuclear cells, the storage of RNA on dried membrane is convenient, and the membranes are easily transported to test/research facilities from the clinical sites. We hope this methodology encourages physician scientists to bring more clinical specimens into research fields to bridge between basic science and clinical medicine.
References
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HindIII; lane 1, pellet at day 0;
lane 2, membrane at day 0; lane 3, membrane at
day 7. Lower panel: lane 1, pellet at day 0;
lane 2, membrane at day 0; lane 3, membrane at
day 7; lane M, 100-bp DNA ladder. (B) One
milliliter of ACD blood was mixed with 9 mL of hypotonic solution (50
mmol/L NaCl) and was passed through RiboCap glass fiber syringe
filters. Three milliliters of denaturing solution were applied, and the
pass-through fraction was stored at room temperature for 14 days. RNA
was extracted twice with phenol/chloroform/isoamyl alcohol, followed by
ethanol precipitation. As control experiments, leukocytes were pelleted
by centrifugation, followed by RNA isolation with a standard acid
guanidine procedure (