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Technical Briefs |
1 Laboratory of Applied Molecular Technologies, Center for Human Genetics, Université Catholique de Louvain, Brussels, Belgium;2 Epidemiology and Biostatistics, Clos-Chapelle-aux-Champs, Université Catholique de Louvain, Brussels, Belgium;3 Defense Laboratories Department, Belgian Armed Forces, Brussels, Belgium
aaddress correspondence to this author at: Applied Molecular Technologies, Center for Human Genetics, Clos Chapelle-aux-Champs, 30, UCL/30.46, B-1200 Brussels, Belgium; fax 32-2-764-39-59, e-mail gala{at}lbcm.ucl.ac.be
ß-Thalassemia is a genetic hemolytic disorder characterized by diminished production of ß-globin chains, attributable to alterations within coding and noncoding sequences of the ß-globin gene (1). Whether noncoding mutations are involved in the decrease of ß-globin chain production or are simple polymorphisms remains a difficult issue (2). Although stable transfection with constructs bearing key regulatory ß-locus control region elements upstream of the promoter are commonly used to assess the transcriptional effect of noncoding mutations in ß-globin (3)(4)(5)(6)(7), discrepant results are still reported, as evidenced recently by the research team that used Northern blotting or ribonuclease protection assay to assess the functional impact of the +10 (-T) deletion (3)(5)(8). Although highly accurate, the competitive reverse transcription-PCR (RT-PCR) technique that we recently developed to quantify the transcriptional effect of genetic alterations in any part of the human ß-globin gene is tedious and time-consuming (6). There is a need, therefore, for an expression method that is accurate, rapid, easier to perform, and well controlled quantitatively.
In our current study, we developed a TaqMan® real-time quantitative RT-PCR assay (QRT-PCR), using the same in vitro expression model and the following human ß-globin variants: –223T
C, –101CT, –30TA, +20CT, IVS-I-108TC, +10 (-T), and +156531577 (6). An additional codon 39 stop-mutation variant construct was included as a control that markedly affects human ß-globin mRNA concentrations (4). The ß-globin expression for each construct was quantified by real-time QRT-PCR, and the results were compared with those originally obtained with the competitive RT-PCR (6), except for the wild-type, the +10 (-T), and the +4043 (-AAAC) 5'-untranslated region deletion variant constructs, for which both quantitative methods were performed in parallel on the same total RNA. RNA extraction, reverse transcription, and competitive RT-PCR were carried out as described previously (6).
Gene-specific PCR primers and TaqMan probes for human ß-globin (GenBank accession no. AF007546) and the mouse housekeeping gene glyceraldehyde phosphate dehydrogenase (mGAPDH; GenBank accession no. BC083149) were designed by use of Primer ExpressTM Software (Ver. 1.5; PE Applied Biosystems). The primers and probe for human ß-globin were as follows: sense primer, 5'-TGCACGTGGATCCTGAGAACT-3'; antisense primer, 5'-AATTCTTTGCCAAAGTGATGGG-3'; probe, 5'-CAGCACGTTGCCCAGGAGCCTG-3'. The primers and probe for mGAPDH were as follows: sense primer, 5'-CCAAGGAGTAAGAAACCCTGGA-3'; antisense primer, 5'-CGAGTTGGGATAGGGCCTCT-3; probe, 5'-CACCCACCCCAGCAAGGACACTG-3'. Each probe was labeled with a fluorescent 5' reporter dye [6-carboxyfluorescein (FAM)] and a 3' quencher [6-carboxytetramethylrhodamine (TAMRA)]. mRNA was reverse-transcribed as described previously (6). For each sample of total RNA, 3 separate reverse transcriptions were carried out. For each cDNA, a triplicate amplification was carried out using 2.5 µL of cDNA, 12.5 µL of Universal PCR Master Mix (2x; Applied Biosystems), 300 nM each of the primers, and 100 nM probe in a total reaction volume of 25 µL. TaqMan PCR was performed in a 25-µL total volume containing 12.5 µL of Universal PCR Master Mix (PE Applied Biosystems), 300 nM each of the primers, and 100 nM fluorescent probe. The human ß-globin and mGAPDH genes were amplified in parallel, and the reaction was carried out as described previously (9).
Data were recorded as cycle threshold (Ct) on a TaqMan 7700 Sequence Detection System (Applied Biosystems), using the analytical software from the same manufacturer. The mean Ct value for mGAPDH was subtracted from the mean Ct value for the human wild-type ß-globin. This 
Ct value obtained with a mutated construct was then subtracted from the Ct value obtained with the wild-type construct, giving a Ct value. As amplification efficiencies of the human ß-globin and mGAPDH were comparable (data not shown), the amount of human ß-globin mRNA, normalized to mGAPDH, was given by the relationship 2–Ct. Two other mouse housekeeping genes were compared with mGAPDH: cyclophilin A (CypA; accession no. X52803) and hypoxanthine guanine phosphoribosyltransferase gene (HPRT1; accession no. BC083415). The mGAPDH mean Ct values obtained with the human wild-type ß-globin was normalized alternatively with either housekeeping gene. Data were reported as the mean (SD). For each construct, the ß-globin expression values, as given by the 2–Ct values, were compared by use of the Student t-test. Statistical significance was set to 0.05. All analyses were done with the SPSS Statistical Package, release 12.0 for Windows (SPSS, Inc.).
mGAPDH expression appeared constant when normalized with either the CypA or HPRT1 mouse housekeeping gene and was therefore chosen for all experiments (data not shown). As for the wild-type construct, expression results for constructs +10 (-T) and +4043 (-AAAC) were tested concomitantly with both methods on the same cDNA. This was prompted by the current observation of reversed +10 (-T) and +4043 expression results by QRT-PCR compared with the original results obtained with competitive RT-PCR (6). Sequence analysis of the latter constructs, before concomitant retesting, confirmed that the inversion of the results obtained in the original publication was caused by mislabeling of the tubes containing the respective constructs; it also validated the current data. Compared with the relative expression of the wild-type ß-globin construct, both quantitative methods produced comparable human ß-globin expression results in MEL cells (Table 1
). The only exceptions concerned the –30TA, –101CT, and del +15651577 constructs, for which we observed significant differences (Table 1
). As discussed previously (6), these constructs are notably associated with very low human ß-globin expression, ranging between 30% and 10% of the expression of the wild type, which increases interassay variability. Nonetheless, the QRT-PCR result obtained with the codon 39 stop-mutation variant was comparable to the expression determined with other methods [6 ( (3))% vs 2%, respectively, of the amount of wild-type mRNA accumulated] (4).
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In summary, there was no significant difference between the competitive RT-PCR and the proposed QRT-PCR carried out on the same cDNA, nor were there differences between the original competitive RT-PCR and the QRT-PCR results, except when human ß-globin expression was strongly diminished. QRT-PCR appears as accurate as competitive RT-PCR but is much faster, less labor-intensive, and devoid of molecular carryover. In addition, an identical protocol could be used to quantify, in parallel, the effects of several mutations or deletions in the same 96-well plate. QRT-PCR thus is suitable for quantifying the transcriptional impact of any mutation or deletion introduced by fragment exchange or directed mutagenesis in noncoding regions of the human ß-globin gene.
References
G mutation at 6 bp 3' to the termination codon causes ß-thalassaemia by decreasing the mRNA level. Br J Haematol 2002;118:671-676.[Medline]
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