A competitive reverse transcription–PCR to study apolipoprotein gene expression

Department of Internal Medicine I, University of Heidelberg, Endocrinology and Metabolism, Bergheimer Strasse 58, D-69115 Heidelberg, Germany.
^a Address correspondence to this author at: Medizinische Universitätsklinik Heidelberg, Abteilung Innere Medizin I, Endokrinologie und Stoffwechsel, Bergheimer Strasse 58, D-69115 Heidelberg. Fax 49 6221 56 53 42; e-mail uk691jd{at}genius.embnet.dkfz-heidelberg.de.

	Abstract

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We developed a rapid and simple competitive reverse transcription-polymerase chain reaction for the quantification of apo mRNA in human monocyte-derived macrophages. The method was applied, and its reliability was shown in patients with the familial lipoprotein disorder, type III hyperlipoproteinemia. Type III hyperlipoproteinemic patients express markedly higher concentrations of apo mRNA when compared with healthy controls. Patients with this disease are usually (>90%) homozygous for a receptor binding-defective isoform of apolipoprotein apo E (apo E2). The higher expression of apo mRNA in the patients could, therefore, be a physiological mechanism to compensate for functionally defective apo E. The developed procedure might be valuable in assessment of apo gene expression in human disease.

	Introduction

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Apolipoprotein E (apo E)¹ not only plays an important role in lipid metabolism but also has essential functions in general cell biology (1). Synthesized mainly by the liver, apo E is also found in many other tissues and cells, such as the spleen, lung, smooth muscle, macrophages, and brain astrocytes (2)(3). Additionally, a growing body of evidence has implicated apo E as critical in the pathogenesis of Alzheimer's disease (4). Hence, interest concerning the molecular regulation of apo gene expression is increasing. Accordingly, various methods referring to this topic have been described (5)(6)(7). However, when classical molecular biological techniques are used, problems may arise because radioactive probes as well as large quantities of cells and RNA are needed. Therefore, we developed a new and practical approach for the rapid and reliable quantitation of apo mRNA. Because our procedure is based on a PCR method, only minimal amounts of total RNA are required, enabling us to quantify apo mRNA in cultures of human monocyte-derived macrophages obtained from <50 mL of heparinized blood.

Apo E mediates both the uptake and catabolism of cholesterol- and triglyceride-rich lipoproteins via the LDL receptor and the LDL receptor-related protein (8). Apo E is a single chain polypeptide of 299 amino acids with a calculated molecular weight of 34 200 Da (9). The nucleotide sequences of the human apo gene (10) and mRNA (11) have been determined. The apo gene, which has been mapped to chromosome 19q13.2 (12), consists of four exons and three introns and is a total of 3597 nucleotides in length.

Apo E is genetically polymorphic; three codominant alleles at the apo gene locus (apo2, apo3, and apo4) code for three different apo E isoforms, designated apo E2, apo E3, and apo E4 (13), giving rise to six common apo E phenotypes: three homozygous (apo E4/4, apo E3/3, and apo E2/2) and three heterozygous (apo E4/3, apo E4/2, and apo E3/2). The common apo E2 (Arg₁₅₈ Cys) isoform exhibits a markedly reduced affinity for hepatic lipoprotein receptors (14). Homozygosity for this isoform is a prerequisite for the familial lipoprotein disorder, type III hyperlipoproteinemia (HLP), causing a disturbance of the metabolism of triglyceride- and cholesterol-rich remnants of chylomicrons, VLDLs, and intermediate-density lipoproteins, collectively referred to as ß-VLDLs (15). Affected individuals develop accelerated and premature atherosclerosis involving both coronary and peripheral arteries. We chose monocyte-derived macrophages to investigate apo gene expression in patients with type III HLP and healthy control subjects.

The regulation of apo E protein secretion has been extensively studied, in particular in macrophages (2)(6)(7)(16)(17). However, data concerning apo transcription in this system are rare, mainly because many cells have to be cultivated to obtain adequate mRNA quantities to perform traditional RNA blotting techniques. Making use of a combination of reverse transcription and a specifically designed competitive PCR, we were able to quantify specific apo mRNA transcripts from <1 µg of total RNA. Thus, this method may prove useful for studying gene expression; in particular, it may prove useful when the amount of biological material is very small and high assay sensitivity is required.

	Materials and Methods

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patients and controls
Eleven patients between 31 and 75 years of age (7 men and 4 women) with "classical" type III HLP were selected for the study. As a control group, sex- and age-matched healthy individuals were chosen. All type III HLP patients were homozygous for the apo2 allele, whereas control subjects were of apo3/3 (n = 10) and apo4/3 (n = 1) genotype, as determined by the method of Hixson and Vernier (18).

isolation of human mononuclear cells and monocyte enrichment
After donors had fasted for 12 to 14 h, 50 mL of venous blood with the addition of 10 U heparin/mL was obtained. Blood samples were generally taken in the morning and subsequently submitted to standard Ficoll Hypaque density gradient centrifugation (19). Isolated mononuclear cells were washed with sterile RPMI-1640 medium without added serum (Life Technologies). Monocytes were enriched by allowing the cells (20 x 10 in 10 mL of serum-free medium) to adhere to 25-cm plastic flasks (Costar) for 60 min at 37 °C. After this time, the supernatant was decanted, and nonadherent cells were removed by gently rinsing the flasks with ice-cold serum-free phosphate-buffered saline (pH 7.4) without added calcium or magnesium. Serum-free culture medium (10 mL; Macrophage-SFM, Life Technologies) containing 100 U/mL penicillin and 100 µg/mL streptomycin was added to the adherent cells, and the cells were incubated for 5 days at 37 °C in a humidified atmosphere of 5% CO₂. Cells were harvested by scraping the flasks with a rubber policeman. After the cells were washed with phosphate-buffered saline, the viability exceeded 95%, as determined by trypan blue exclusion. The cell population was composed of >90% CD14-positive cells, as shown by flow cytometry-staining. After centrifugation, cell pellets were lysed in 500 µL of guanidinium thiocyanate buffer and stored at -80 °C until RNA preparation.

rna preparation
Total cellular RNA was extracted from guanidine thiocyanate-lysed cells according to a modified method of Chomczynski and Sacchi (20), using a commercially available RNA isolation kit (Fluka). RNA pellets were dissolved in 10–20 µL of diethyl pyrocarbonate-treated water, and aliquots were taken to measure optical density and to check RNA integrity by denaturing agarose gel electrophoresis (21). RNA samples were also stored at -80 °C.

reverse transcription
Purified total RNA was reversely transcribed using avian myeloblastosis virus reverse transcriptase obtained with the Superscript(TM) Preamplification System for First Strand cDNA Synthesis (Life Technologies). As a general rule, 1 µg of total RNA was used for cDNA synthesis, which was performed in a sample volume of 20 µL, according to the manufacturer's instructions and using oligo(dT) (12–18 mer) as a primer. Finally, reversely transcribed samples were adjusted with water to a final volume of 100 µL and stored at -80 °C.

pcr
PCR was performed using thin-walled PCR tubes in a Minicycler(TM) (MJ Research). Reactions were performed in a total volume of 50 µL containing final concentrations of 20 mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 5 mmol/L MgCl₂, 0.2 mmol/L of each dNTP, 0.6 µmol/L of each primer, and 2 U of Taq polymerase (Life Technologies). Reactions were started with an initial step for denaturation at 94 °C for 3 min, followed by cycles for denaturing at 94 °C for 45 s, annealing at 55 °C for 45 s, and DNA synthesis at 72 °C for 90 s. After the last cycle, reactions were continued at 72 °C for 10 min for primer extension. The numbers of cycles were 35 for apo-specific reactions and 30 for glyceraldehyde-3-phosphate dehydrogenase-specific (GAPDH-specific) PCR, respectively. Primers were synthesized by MWG-Biotech. The following apo-specific primers were used (22): 5'-TTC CTG GCA GGA TGC CAG GC-3' (5'-primer, 20-mer) and 5'-GGT CAG TTG TTC CTC CAG TTC -3' (3'-primer, 21-mer). GAPDH-specific PCR was performed using the following primers (23): 5'-TGA AGG TCG GAG TCA ACG GAT TTG GT-3' (5'-primer, 26-mer) and 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3' (3'-primer, 24-mer).

synthesis of an internal dna standard and competitive pcr
For synthesis of the internal apo DNA standard, the 270-bp PCR apo fragment served as a template. For this purpose, the DNA band was cut out of the agarose gel. The gel slice was cut into small pieces and incubated for several hours in 0.5 mL Tris-acetate buffer (pH 7.4) at room temperature. A few microliters of the supernatant were used in a second PCR, which was performed using the same 5'-primer but replacing the original 3'-primer with an internal 3'-linker primer of the following sequence: 5'-GGT CAG TTG TTC CTC CAG TTC TTG GGT GAC TTG-3' (33-mer). The resulting 216-bp amplification product was again isolated from the agarose gel and reamplified in several PCR samples using the original 5'-primers and 3'-primers, respectively, to obtain sufficient DNA, which was subsequently purified from agarose gels using a modification of the method of Vogelstein and Gillespie (24). After the optical density of the samples was measured, aliquots of standard DNA were made that contained 1 pmol/L 216-bp fragment and 50 mg/L glycogen for stabilization. Aliquots were stored at -20 °C until they were used in competition experiments.

For competitive apo PCR, 4 µL of cDNA mixture was placed in each of 6–8 PCR test tubes. Internal standard DNA was added to the tubes in increasing amounts, generally in a range of 0.03–1.6 pmol/L. After amplification, samples were electrophoresed in a Tris-borate buffer system using 2% agarose gels containing 0.36 mg/L ethidium bromide. DNA bands were visualized under UV light and detected with a digitalizing gel documentation system (INTAS). Quantification was performed using the NIH Image 1.4.4. computer system.

For competitive GAPDH-PCR, the cDNA mixture was diluted 1:25 before use in the experiments. Samples were then prepared in a manner similar to that described for apo. The 983-bp amplification product obtained using the GAPDH-specific primers competed with an internal DNA standard (Clontech), yielding a 630-bp PCR product.

statistical analysis
Statistical analysis was performed with the Mann–Whitney U-test for nonparametric data. P values <0.05 were considered significant.

	Results

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In our approach to the quantification of apo mRNA, two essential steps of nucleic acid analysis have been combined. In the first step, total RNA–including mRNA–is reversely transcribed to yield a cDNA. In the second step, this cDNA is analyzed for apo-specific transcripts by use of a highly sensitive PCR. This method, therefore, is commonly referred to as reverse transcription-PCR (RT-PCR). However, because of the exponential nature of PCR, small variations in amplification efficiency can lead to dramatic changes in product. Thus, quantitative information may be difficult to obtain. Therefore, we have designed an internal DNA standard that shares the same primer sites as the target transcript. This DNA may be coamplified simultaneously in each PCR tube, thus allowing, by means of competition, an exact quantification of the target transcript (competitive RT-PCR) (25)(26). Because of this competitive design, the molar ratio of the target-to-standard sequence remains constant throughout the amplification, not only during the exponential but also during the nonexponential phase of the reaction, rendering this technique independent of both the number of cycles (27) and other so-called "tube-to-tube" effects.

For apo amplification, we chose a pair of primers previously described by Wang et al. (22) that yielded a 270-bp amplification product. Although the annealing site for the 3'-primer (5'-GGT CAG TTG TTC CTC CAG TTC-3') is located completely within the fourth exon of the apo gene (nucleotides 3640–3660), the 5'-primer (5'-TTC CTG GCA GGA TGC CAG GC-3') anneals to the last 10 nucleotides of the second exon and the first 10 nucleotides of the third exon (nucleotides 1717–1726 and 2920–2929, respectively). Therefore, its annealing site is only formed in the correctly spliced mRNA. As expected, we did not obtain any amplification products when we used genomic DNA or total RNA that had not been reversely transcribed before PCR.

For internal standard generation, we chose a method previously described by Förster (28), which is shown in a schematic view in Fig. 1 . For this purpose, we synthesized a 33-mer 3'-linker primer (5'-GGT CAG TTG TTC CTC CAG TTC TTG GGT GAC TTG-3') that anneals to a 18-bp sequence at nucleotides 184–201 of the 270-bp fragment and that carries an overhanging 5'-tail complemetary to the original 3'-primer annealing site. Because the original 3'-primer site is 21 nucleotides in length, one would expect the linker primer to consist of 39 nucleotides. However, a 33-mer primer was sufficient because the last six matching nucleotides of its 3'-annealing site were identical with those beginning the complementary sequence of the original 3'-primer. Using the 270-bp fragment as a template in a reaction in which the original 3'-primer had been replaced by this 3'-linker primer, PCR generated a DNA fragment that was shorter in length (216 bp) but was, apart from the deleted nucleotides, identical to the 270-bp fragment with respect to sequence and primer sites and was, therefore, suitable for use as a competitive standard.

View larger version (15K): [in this window] [in a new window]   Figure 1. Schematic view of internal standard generation. PCR 1 was performed using the original 5'-primer and 3'-primer (22), generating a 270-bp amplification product from apo E cDNA. PCR 2 was performed with the 270-bp fragment as a template and using the original 5'-primer and a specially designed 3'-linker primer (25), generating a 216-bp DNA fragment that could be reamplified in PCR 3, using the original 5'-primer and 3'-primer.

A typical competitive PCR experiment in which a series of samples were amplified using the original 5'- and 3'-primers is shown in Fig. 2 A(22). Samples each contain the same amount of cDNA mixture but contain increasing amounts of the 216-bp fragment as a competitive internal standard. After gel electrophoresis, band intensities were determined using a computer imaging system, and data were plotted as log I_{internal standard} x k/I_target (I, intensity of the band; k, correction factor) vs log concentration of added internal standard (Fig. 2B ; because the same molar amount of a longer DNA fragment will stain more intensely than a shorter one, the intensity values of the internal standard must be corrected with respect to the molar ratio of the two fragments using the correction factor k, which is 270 bp/216 bp = 1.25). The amount of apo-specific cDNA, which reflects the amount of mRNA, can now be calculated from the plot at the point where identical molar amounts of both fragments are obtained (log I_{internal standard} x k/I_target = 0) (Fig. 2B ).

View larger version (22K): [in this window] [in a new window]   Figure 2. Competitive PCR for quantification of apo-specific cDNA. (A) Gel electrophoresis of samples 1–6, each containing 4 µL of cDNA and increasing amounts (0.07, 0.1, 0.3, 0.6, 1.0, and 1.6 amoles, respectively) of the internal 216-bp standard DNA for competition. (B) Plot of log Iinternal standard x k/Itarget (I, intensity of band; k, correction factor) vs log concentration of the added internal standard. The amount of apo-specific cDNA is calculated at the point where identical molar amounts of both amplification products are obtained (log Iinternal standard x k/Itarget = 0).

Apo mRNA data obtained for different samples of cDNA by the described method are only comparable under the assumption that the efficiency of cDNA synthesis was identical in each tube. This, however, is rather unlikely because very often tube-to-tube effects are observed in this procedure. Therefore, all cDNA samples were additionally analyzed with respect to GAPDH gene expression, which is known to be an almost constantly expressed "housekeeping" gene and often is used as a molecular standard (23). Data are thus presented as moles of apo mRNA per mole of GAPDH mRNA. In terms of validation of our method, we have performed several experiments, one of which is shown in Table 1 . Intraassay variation was low and reproducibility of data was high, with SDs in a range of <15% of the mean.

View this table: [in this window] [in a new window]   Table 1. Method validation: ratio of apo and GAPDH mRNA in dependence of reversely transcribed total RNA.

Making use of the described PCR method, we analyzed the apo gene expression in macrophages of 11 patients with classical type III HLP and homozygosity for apo E2 (Arg₁₅₈ Cys). More than 90% of the patients with this familial lipoprotein disorder are homozygous for this apolipoprotein isoform, which exhibits markedly reduced binding to hepatic lipoprotein receptors (14). Reduced receptor affinity may, therefore, be compensated for by higher serum concentrations of this protein. In our study, we used macrophages because they are easy to obtain and have been shown to secrete essential amounts of plasma apo E (29). We showed that macrophages of patients with type III HLP express a markedly higher amount of the apo gene (0.57 ± 0.34 moles/mole GAPDH) when compared with sex- and age-matched controls (0.30 ± 0.17 moles/mole GAPDH) (P <0.05) (Table 2 and Fig. 3 ).

View this table: [in this window] [in a new window]   Table 2. Apo gene expression in patients with type III HLP compared with a control group of healthy subjects (means ± SD).

View larger version (17K): [in this window] [in a new window]   Figure 3. Box and whisker plot of apo mRNA concentrations in patients with type III HLP compared with a control group of healthy subjects. Boxes indicate both medians and the 25th and 75th percentiles, respectively. Whiskers indicate maximum and minimum values.

	Discussion

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Until now, most studies concerning gene expression made use of traditional techniques such as Northern and "dot blot" hybridization, which require large quantities of cells and mRNA, respectively. Moreover, these methods permit only crude quantitation of mRNA. Therefore, the combination of reverse transcription with the high sensitivity of PCR has been increasingly applied to amplifying cDNA copies of low abundance mRNA. In particular, when combined with competitive internal DNA standards, this procedure is the method of choice for detecting and quantifying minimal amounts of mRNA transcripts (25)(26)(27). As reported here, our approach with respect to the analysis of apo gene expression enables us to detect far less than one attomole of apo-specific sequences. This may be achieved by the use of a cDNA prepared from <1 µg of total RNA, which can be obtained from as few as 3 x 10 macrophages. In contrast, Northern blot analysis was performed in monocyte-derived macrophages by making use of ~10–20 x 10 cells and as much as 10–20 µg of total RNA (30). The method of apo mRNA quantification presented here should, therefore, prove particularly useful in cases where cells or mRNA are in low concentrations or difficult to obtain.

As we expected, apo gene expression is relatively high in human monocyte-derived macrophages. Thus, in cells of healthy subjects apo mRNA concentrations are ~30% with respect to the expression of the common housekeeping gene GAPDH. In macrophages of patients with type III HLP, apo gene expression is nearly twice as high. These results are in accordance to data on serum concentrations of apo E, which have also been reported to be nearly twice as high in patients with apo2/2 genotype when compared with a control group with apo3/3 genotype (31). One could speculate, therefore, that higher apo E serum concentrations in type III HLP patients might be due to increased expression of the defective gene product rather than to reduced clearance from plasma. It would also be interesting to confirm our findings by using in vivo macrophages or in experiments using human liver tissue, which, however, is difficult to obtain.

	Footnotes

 
¹ Nonstandard abbreviations: apo E, apolipoprotein E; HLP, hyperlipoproteinemia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; and RT-PCR, reverse transcription-PCR.

	References

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1. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 1988;240:622-630. [Abstract/Free Full Text]
2. Zannis VI, Cole FS, Jackson CL, Kurnit DM, Karathanasis ST. Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte macrophages. Biochemistry 1985;24:4450-4455. [Medline] [Order article via Infotrieve]
3. Elshourbagy NA, Liao WS, Mahley RW, Taylor JM. Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of the rats and marmosets. Proc Natl Acad Sci U S A 1985;82:203-207. [Abstract/Free Full Text]
4. Weisgraber KH, Roses AD, Strittmatter WJ. The role of apo-lipoprotein E in the nervous system. Curr Opin Lipidol 1994;5:110-116. [Medline] [Order article via Infotrieve]
5. Diedrich JF, Minnigan H, Carp RI, Whitaker JN, Race R, Frey R, et al. Neuropathological changes in scrapie and Alzheimer's disease are associated with increased expression of apolipoprotein E and cathepsin D in astrocytes. J Virol 1991;65:4759-4768. [Abstract/Free Full Text]
6. Auwerx JH, Deeb S, Brunzell JD, Peng R, Chait A. Transcriptional activation of the lipoprotein lipase and apolipoprotein E genes accompanies differentiation in some human macrophage-like cell lines. Biochemistry 1988;27:2651-2655. [Medline] [Order article via Infotrieve]
7. Basheeruddin K, Rechtoris C, Mazzone T. Transcriptional and post-transcriptional control of apolipoprotein E gene expression in differentiating human monocytes. J Biol Chem 1992;267:1219-1224. [Abstract/Free Full Text]
8. Kowal RC, Herz J, Goldstein JL, Esser V, Brown MS. Low density lipoprotein receptor-related protein mediates uptake of cholesterol esters derived from apoprotein E-enriched lipoproteins. Proc Natl Acad Sci U S A 1989;86:5810-5814. [Abstract/Free Full Text]
9. Rall SC, Jr, Weisgraber KH, Mahley RW. Human apolipoprotein E: the complete amino acid sequence. J Biol Chem 1982;257:4171-4178. [Free Full Text]
10. Paik YK, Chang DJ, Reardon CA, Davis GE, Mahley RW, Taylor JM. Nucleotide sequence and structure of the human apolipoprotein E gene. Proc Natl Acad Sci U S A 1985;82:3445-3449. [Abstract/Free Full Text]
11. McLean JW, Elshourbagy NA, Chang DJ, Mahley RW, Taylor JM. Human apolipoprotein E mRNA: cDNA cloning and nucleotide sequencing of a new variant. J Biol Chem 1984;259:6498-6504. [Abstract/Free Full Text]
12. Das HK McPherson J, Bruns GAP, Karathanasis SK, Breslow JL. Isolation, characterization, and mapping to chromosome 19 of the human apolipoprotein E gene. J Biol Chem 1985;260:6240-6247. [Abstract/Free Full Text]
13. Zannis VI, Breslow JL. Human very low density apolipoprotein E isoprotein polymorphism is explained by genetic variation and post-translational modification. Biochemistry 1981;20:1033-1041. [Medline] [Order article via Infotrieve]
14. Schneider WJ, Kovanen PT, Brown MS, Goldstein JL, Utermann G, Weber W, et al. Familial dysbetalipoproteinemia: abnormal binding of mutant apolipoprotein E to low density lipoprotein receptors of human fibroblasts and membranes from liver and adrenals of rats, rabbits and cows. J Clin Invest 1981;68:1075-1085.
15. Mahley RW, Rall SC, Jr. Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited disease 7th ed. 1995:1953-1980 McGraw-Hill New York. .
16. Brown MS, Goldstein JL. Apoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983;52:223-261. [ISI][Medline] [Order article via Infotrieve]
17. Kayden KJ, Maschio F, Traber MG. The secretion of apolipoprotein E by human monocyte-derived macrophages. Arch Biochem Biophys 1985;239:388-395. [ISI][Medline] [Order article via Infotrieve]
18. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hha I. J Lipid Res 1990;31:545-548. [Abstract]
19. Böyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1g. Scand J Clin Lab Invest 1948;97:77-89.
20. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. [ISI][Medline] [Order article via Infotrieve]
21. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a ulaboratory manal, 2nd ed. Plainview: Cold Spring Harbor Laboratory Press, 1989..
22. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A 1989;86:9717-9721. [Abstract/Free Full Text]
23. Tso JK, Sun XH, Kao TH, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 1985;13:2485-2502. [Abstract/Free Full Text]
24. Vogelstein B, Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A 1979;76:615-619. [Abstract/Free Full Text]
25. Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A 1990;87:2725-2729. [Abstract/Free Full Text]
26. Siebert PD, Larrick JW. PCR-MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR. Biotechniques 1993;14:244-249. [ISI][Medline] [Order article via Infotrieve]
27. Cottrez F, Auriault C, Capron A, Groux H. Quantitative PCR: validation of the use of a multispecific internal control. Nucleic Acids Res 1994;22:2712-2713. [Free Full Text]
28. Förster E. An improved general method to generate internal standards for competitive PCR. Biotechniques 1994;16:18-20. [ISI][Medline] [Order article via Infotrieve]
29. Driscoll DM, Getz GS. Extrahepatic synthesis of apolipoprotein E. J Lipid Res 1984;25:1368-1379. [Abstract]
30. Haskill S, Johnson C, Eierman D, Becker S, Warren K. Adherence induces selective mRNA expression of monocyte mediators and proto-oncogenes. J Immunol 1988;140:1690-1694. [Abstract]
31. Utermann G. Genetic polymorphism of apolipoprotein E: impact on plasma lipoprotein metabolism. Crepaldi G Tiengo A Baggio G eds. Diabetes, obesity and hyperlipidemias-III 1985:1-28 Excerpta Medica Amsterdam. .