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Technical Briefs |
1
INSERM U90 and Clin. Biochem. Lab., CHU Necker, 149 rue de Sèvres, 75015 Paris, France;
2
Mol. Chem. Lab., Saint-Luc Clin. Univ., Clos Chapelle aux Champs 30, Bruxelles, Belgium;
3
Urol. Unit and Exp. Surg. Lab., CHU Bicêtre, 78 rue du Général Leclerc, 94270 Le Kremlin Bicêtre, France;
a address correspondence to this author, at: Lab. de Biochim. A, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France
Although not in routine use, molecular techniques appear able to detect small numbers of solid tumor cells in blood, lymph nodes, and bone marrow (for review see 1). Provided that a given mRNA is expressed exclusively in tumor tissue and not by nucleated blood cells, lymph node cells, or bone marrow cells, its presence outside the organ identifies disseminated metastatic cells. In prostate cancer (CaP), early detection of metastases by molecular staging could help in selecting which patients would benefit most from surgical therapy (2).
Several reverse transcriptase-polymerase chain reaction (RT-PCR) protocols are able to amplify the mRNAs encoding prostate-specific antigen (PSA) or prostate-specific membrane antigen (PSMA) in blood (1), lymph nodes (3), or bone marrow (4). Nevertheless, the results reported have varied so widely (e.g., positive PSA RT-PCR results ranging from 25% (5) to 80% (6) for metastatic CaP patients) that definitive conclusions about the clinical relevance of this new molecular tool have not yet been established. The heterogeneity of both preanalytical and analytical steps of PCR protocols may explain the discrepancies observed between studies. To our knowledge, no systematic studies of the successive steps of the RT-PCR method have yet been performed. To better standardize the assay, we have assessed the effects of the storage conditions of blood samples and delay before processing.
To compare the effect of delayed vs immediate sample processing, we collected 250-mL blood samples in 25 10-mL EDTA-treated tubes (Becton Dickinson) from 4 apparently healthy blood donors. Each sample was then supplemented with 10 metastatic lymph-node-derived prostate carcinoma LNCaP cells (7), and gently mixed. One tube of supplemented sample from each donor was processed immediately after preparation to serve as a positive control; the remaining ones were tested with various storage temperatures and delays. Five were sealed and left at room temperature (19–22 °C) for 3, 6, 9, 12, or 24 h. Eighteen, 9 refrigerated immediately at 4 °C and another 9 mixed first with 2 volumes of NH4Cl (9 g/L) before cooling to 4 °C, were left for 6 or 12 h, or 1, 2, 3, 6, 9, 12, or 15 days at the same temperature. Each blood sample was processed by the same protocol, as follows: Plasma was discarded and the remaining blood cells were diluted in 2 volumes of the NH4Cl solution and shaken overnight at 4 °C. Each sample was then centrifuged at 1000g for 20 min and the supernatant removed. Total RNA was extracted from these cells by the guanidium-thiocyanate/phenol/chloroform extraction technique (8). The RNA pellet obtained after ethanol/sodium acetate precipitation was dried under reduced pressure and dissolved in 50 µL of RNase-free water; the yield of RNA was quantified by spectrophotometric analysis at 260 nm. RNA quality was evaluated by examining the average ratio of UV absorbance at 260 and 280 nm and then checked for purity by agarose gel analysis.
RT-PCR for PSA or PSMA was performed as previously described (9), with normalization by amplification of ß-globin (10). Briefly, 1 µg of total RNA was used for synthesis of the first strand of cDNA with SuperScript II reverse transcriptase and 100 ng oligo(dT)l2–18 as template (Life Technologies). Then, 1 µL of cDNA was amplified by PCR by the following protocol: 94 °C for 1 min (2 min for the first cycle); 62 °C, 60 °C, and 58 °C for 1 min (for PSA, PSMA, and ß-globin, respectively); and 72 °C for 1 min (10 min for the last cycle) for 25 cycles. The PCR products (15 µL) were run on ethidium bromide-stained agarose gels (20 g/L) and visualized by transillumination. Fluorescence was captured with a CCD camera and integrated by use of NIH Image 1.7 software. Statistical analysis of the results of three independent experiments performed in duplicate was done with GraphPad Prism/Stat(TM) software.
Room temperature (19–23 °C) storage of blood dramatically affected
the RNA. Within 6 h, there was an important loss of RNA as
indicated by UV quantification (Fig. 1
A) and confirmed by gel analysis (not shown). On samples stored
from 6 to 24 h at room temperature, amplification of PSA and PSMA
cDNAs was still possible but was impaired, as indicated by a decrease
in the intensity of bands for PCR products (Fig. 1
, B and C).
Extracting RNA from the 9 samples cooled immediately to 4 °C gave
comparable signals to those for samples stored 6 h to 6 days at
that temperature. Amplification of both PSA (Fig. 1B
) and PSMA (Fig. 1C
) cDNAs also gave comparable results for the samples stored 6 h
to 6 days. The decrease in PSMA cDNA signals in the samples stored for
12 or 15 days at 4 °C apparently paralleled the decrease in PSA
amplification and reflected loss of RNA after storage for >6 days. The
immediate addition of ammonium chloride did not enhance the apparent
stability of RNA, but PSMA and PSA amplifications were slightly lower
in NH4Cl conditions than in the standard 4 °C
storage after 6 days or longer. This suggests a long-term inhibitory
effect of NH4Cl on reverse transcriptase: (a)
RNA amounts extracted were identical in the two groups of samples
stored at 4 °C, regardless of NH4Cl addition, and
(b) no discrepant results were observed between PSA and PSMA
cDNAs.
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The current study demonstrates an important loss in RNA recovery within 6 h when samples are stored at room temperature. Stable results for at least 6 days are obtained when samples are immediately stored at 4 °C after sampling. The length of the delay is of particular interest to laboratories receiving and processing blood samples stored for the day or sent at room temperature from remote institutions. Although the cultured LNCaP cells used here may not exactly mimic the behavior of prostate cancer cells, much less other types of cancer cells, our results support the following recommendations: 1) Place blood samples at 4 °C immediately after drawing. 2) Avoid shipping samples to the laboratory at room temperature. 3) If the RNA is to be extracted within 2 weeks, store blood samples immediately at 4 °C without ammonium chloride.
Acknowledgments
This study was supported in part by grants from Association de Recherche contre le Cancer (ARC 1367) and Association de Recherche sur les Tumeurs de la Prostate (ARTP).
Footnotes
References
The following articles in journals at HighWire Press have cited this article:
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  P. Berteau, F. Dumas, J.-L. Gala, P. Eschwege, B. Lacour, M. Philippe, and S. Loric Molecular Detection of Circulating Prostate Cells in Cancer II: Comparison of Prostate Epithelial Cells Isolation Procedures Clin. Chem., August 1, 1998; 44(8): 1750 - 1753. [Full Text] [PDF]
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