Serial competitive RT-PCR using multiple standards.

PCR is widely used for the amplification of DNA and reverse-transcribed RNA. In recent years, real-time PCR has been introduced as a new technique for mRNA quantitation. However, because of the high cost of real-time PCR equipment and supplies, especially using custom-made probes, quantitative RT-PCR is a valuable alternative method. Several quantitative PCR protocols describe the use of a single competitive standard (2,6). The aim of the present study was the development of a protocol using several competitive standards of different sizes to quantify gene expression in a one-tube-amplification procedure. As a model system, endothelin receptor B mRNA was quantified in human endothelial cells. Endothelin-1 is the most potent vasoconstrictor known to date and mediates its effects in cardiovascular physiology and pathophysiology via binding to endothelin receptors type A or B (ETA and ETB) (4). Endothelial cells express ETB receptors only (3,5). Total RNA from primary cultures of human umbilical vein endothelial cells was isolated as previously described (7). A 702-bp fragment of human ETB was cloned from endothelial RNA by RT-PCR (5). Four internally deleted standards (648, 528, 438, and 355 bp) were constructed using linker primer PCR (2). Using this approach, target and standards contain the same sense and antisense primer binding sites. We used following primers: ETB sense: 5′-CGAGCTGTTGCTTCTTGGAGTAG-3′, ETB antisense: 5′-ACGGAAGTTGTCATATCCGTGATC-3′, linker 1: 5′-CTTGGAGTAGGGTGGTCTCTGTGGTTCTGG-3′, linker 2: 5′-CTTGGAGTAGGCAGTTTTACAAGACAGCAA-3′, linker 3: 5′-CTTGGAGTAGGACCTGTGAAATGTTGAGAA-3′, and linker 4: 5′-CTTGGAGTAGCCGTCTTTTGCCTGGTCCTT-3′. Standards were cloned into pCR-Script Amp SK+ Cloning Vector (Stratagene, La Jolla, CA, USA). Standard identity was confirmed by cycle sequencing using ABI PRISM® Dye Terminator Cycle Sequencing Ready Reaction Kit on an automated ABI 373A DNA Sequencer (both from Applied Biosystems, Foster City, CA, USA). Database searches of GenBank® were performed using BLASTN (1). In vitro transcription of cDNA standards was performed using the RNA Transcription Kit (Stratagene), and cRNA was quantified spectrophotometrically at 260 nm. Standard RT after 3 min at 70°C was done for 1 h at 42°C using cRNA random hexamer primers and SuperScript II RNase HReverse Transcriptase (Invitrogen, Paisley, UK). Afterwards, one-fifth of each RT reaction was amplified by PCR with 20 pmol ETB sense and antisense primers: 1 min at 95°C, 1 min at 69°C, and 1 min at 72°C. PCR fragments were separated by agarose gel electrophoresis, stained with ethidium bromide, and documented by photography using Polaroid® film type 665. The absorbances of sample specific target and standard copies were estimated by a personal densitometer (Molecular Dynamics, Sunnyvale, CA, USA). Densities of PCR fragments were normalized with a correction coefficient and logarithm of quotient of samplespecific and standard PCR fragment density graphically plotted versus the amount of standard RNA. With our serial RT-PCR using multiple standards, a one-tube reaction is sufficient to measure the target copy number (Figure 1). For this purpose, a standard mixture including all four standards was used. In this mixture, the highest copy number was assigned to the smallest standard. Each larger standard was added in a 1:2 dilution to the next smaller standard. This standard mixture was diluted and added to a constant amount of total RNA (250 ng) from human endothelial cells. After RT-PCR and gel electrophoresis, the copy number of the target gene was calculated. Using this approach, target and standard mixtures of four different internally deleted standards can be transcribed into cDNA and subsequently amplified at the same rate. The ETB copy number in human endothelial cells was determined using the serial RT-PCR method using multiple standards (x± SEM/μg RNA: 1917.8 ± 36.5; n = 4). The method is highly reproducible. To test the validity of the approach, we compared the well-established competitive approach and our new serial RT-PCR using multiple standards (Figure 2A). An identical copy number of target and standard cDNA was used in each approach. Both competitive and serial RT-PCR using multiple standards were carried out in a parallel manner. We found no significant difference in deviation between the two approaches. In both cases, a difference of approxiBenchmarks