The United States and Canadian Academy of Pathology Lippincott Williams and Wilkins publishes Laboratory Investigation

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		A Rapid, Simple, and Inexpensive Step Facilitates RNA Extraction from Whole Blood Cells
		Orietta Gandini, Francesco Saverio Celi, Massimo Magnanti, Paola Gazzaniga, Ida Silvestri, Barbara Conti, Laura Giuliani, Daniela Mentuccia, and Anna Maria Aglianò
		Dipartimento di Medicina Sperimentale e Patologia, Universita' degli Studi di Roma "La Sapienza," Roma, Italy
		In the present article, we describe a rapid and inexpensive method to separate nucleated cells from whole blood. This step facilitates the subsequent procedures of nucleic acid extraction without interfering with the final method sensitivity. Serial dilutions of HeLa cells were added to peripheral blood samples. Nucleated cells were separated from erythrocytes by adding succinyl-linked gelatin, a plasma expander, to the sample. After RNA extraction by Trizol LS (Life Technologies, Italy), we performed reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting epithelial growth factor-receptor (EGF-r) mRNA, which is expressed in HeLa cells but not in leukocytes. The sensitivity of this procedure was 5 HeLa cells/ml of blood. The use of succinyl-linked gelatin is a step for the isolation of nucleated cells from peripheral blood that allows significant savings in expensive materials. The detection of tumor cell mRNA transcripts in peripheral blood affects the prognostic and therapeutic future of patients with solid tumors. RT-PCR, because of its high sensitivity, is the method of choice, being capable of detecting 10-6 cells/ml in samples from blood or bone marrow of cancerous patients (Raj et al, 1998). Many methods have been suggested to separate nucleated cells from whole blood because the removal of erythrocytes results in a higher yield of RNA and better amplification, given that hemoglobin degradation products, such as hematin and hemin, are responsible for Taq Polymerase inhibition (Klein et al, 1997). In previous studies density gradient centrifugation through Ficoll has been utilized to separate mononuclear cells from blood before RNA extraction by different methods (Dearnaley et al, 1981; Zippelius et al, 1997). Others proposed the use of magnetic antibody-coated beads for the separation of circulating cancer cells (Luke and Kaul, 1998). Alternatively, some authors have used rapid RNA extraction reagents, such as Trizol-LS, RNA-STAT-50, and Ultraspec-3, on whole peripheral blood (Chadderton et al, 1997). Their main conclusion was that Trizol-LS was more consistent in obtaining a pure and sufficient quantity of RNA, with removal of heparin as an obligatory step. However these methods are expensive and require a significant amount of material to obtain high RNA yield. Gelatins have been used for separation of nucleated cells from red blood cells of human umbilical cord blood (Pick et al, 1998). In this procedure a volume of gelatin (1:1) is mixed into the blood sample, and the red cells are allowed to settle by gravity under visual monitoring. The supernatant is then collected for the subsequent cell separation steps. A similar procedure, based on the use of dextran, was applied to separate white blood cells from bone marrow (Johnston et al, 1992). To our knowledge no information is available on the use of gelatins for the separation of nucleated cells for subsequent nucleic acid extraction. We tested succinyl-linked gelatin (0.4% succinylgelatin, 140 mM NaCl, 4.7 mM CaCl2, pH 7.0-7.4; EUFUSIN, Clarmed, Milano, Italy), an inexpensive plasma expander, for the separation of nucleated cells from peripheral blood before the Trizol LS RNA extraction procedure. HeLa cells (1, 10, 50, 100, and 1000 cells) were added to 2 ml of peripheral blood collected in heparin-coated tubes by forearm venipuncture from healthy volunteers. After plasma removal, 1 volume of succinyl-linked gelatin was gently mixed and incubated at room temperature for 30 minutes. The upper clear phase was collected and centrifuged for 5 minutes at 500 X g at room temperature. The pellet was then used for RNA extraction by adding 750 ul of Trizol-LS and then proceeding according to the manufacturer's guidelines. No nucleated cells were observed in the lower phase after erythrocytes lysis. The quality of the RNA obtained was tested by absorbance at 260 and 280 nm. The average yield was approximately 20 ug of RNA/ml of blood, similar to the yield expected by using Trizol-LS on whole blood. Total RNA (1 ug) was subjected to reverse transcription in a final volume of 20 ul with 100 pmol of random examer. Aliquots corresponding to 100 ng of RNA were then amplified using [beta]-actin- and EGF-r-specific primers. EGF-r is known to be expressed on epithelial cells but not in normal mononuclear blood cells (Leitzel et al, 1998). In our hands, the sensitivity of such a method was 5 Hela cells/ml of blood (Fig 1). This RNA extraction method, based on succinyl-linked gelatin utilization, results in a high yield, a rapid time of execution, and a very low cost because of the use of minimal amounts of Trizol-LS on the cellular pellet. We stress that guidelines for the use of Trizol-LS in RNA extraction from blood recommend that blood samples must be diluted 1:1 in H2O and that the ratio of reagent to sample must always be 3:1. Thus, under optimal experimental conditions, RNA extraction from 2 ml of blood would require the use of at least 12 ml of Trizol-LS solution, 16-fold the amount of reagent needed after the succinyl-linked gelatin nuclear cell separation step. Furthermore, because no use of a cell-specific antibody is required, this method represents a versatile tool for the separation of different cell types. In conclusion, the use of "succinyl-linked gelatin" for isolation of nuclear cells from blood is a rapid, technically simple, and inexpensive step that allows a substantial reduction of expensive material. Additional advantages include the absence of heparinase treatment and the need for only a microcentrifuge throughout the entire process, thus increasing the capability of performing a molecular diagnostic assay. References Chadderton T, Wilson C, Bewick M, Gluck S (1997). Evaluation of three rapid RNA extraction reagents: relevance for use in RT-PCR's and measurement of low level gene expression in clinical samples. Cell Mol Biol 43:1227-1234. Dearnaley DP, Sloane JP, Ormerod MG, Steele K, Coombes RC, Clink HM, Powels TJ, Ford HT, Gazet JC, Neville AM (1981). Increased detection of mammary carcinoma cells in marrow smears using antisera to epithelial membrane antigen. Br J Cancer 44:85-90. Klein A, Barsuk R, Dagan S, Nusbaum O, Shouval D, Galun E (1997). Comparison of methods for extraction of nucleic acids from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences. J Clin Microbiol 35:1897-1899. Leitzel K, Lieu B, Curley E, Smith J, Chinchilli V, Rychlik W, Lipton A (1998). Detection of cancer cells in peripheral blood of breast cancer patients using reverse transcription-polymerase chain reaction for epidermal growth factor receptor. Clin Cancer Res 4:3037-3043. Luke S and Kaul K (1998). Detection of breast cancer cells in blood using immunomagnetic bead selection and reverse transcription-polymerase chain reaction. Mol Diagn 3:149-155. Johnston JJ, Boxer LA, Berliner N (1992). Correlation of messenger RNA levels with protein defects in specific granule deficiency. Blood 80:2088-2091. Pick M, Nagler A, Grisaru D, Eldor A, Deutsch V (1998). Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two-step separation procedure. Br J Haematol 103:639-650. Raj GV, Moreno JG, Gomella LG (1998). Utilization of polymerase chain reaction technology in the detection of solid tumors. Cancer 82:1419-1442. Zippelius A, Kufer P, Honold G, Kollermann MW, Oberneder R, Schlimok G, Riethmuller G, Pantel K (1997). Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 15:2701-2708. Received August 12, 1999