Relative Quantification of Cytochrome P450 1B1 Gene Expression in Peripheral Leukocytes Using LightCycler

Materials and Methods

A 10 ml peripheral blood sample from a smoker, A, and a never-smoker, B, were collected and the total RNA was isolated by the Trizol® method. This procedure is an improvement of the single-step method reported by Chomczynski and Sacchi (5) for total RNA isolation and is based on a guanidine-thiocyanate-phenol-chloroform extraction. After DNAse treatment, the total RNA was precipitated. At this step, it is very important to confirm that the isolated total RNA is free of any DNA, since DNA can adulterate the results of the subsequent quantitative PCR. Therefore, the RNA was amplified by regular PCR, for example with β-actin primers, to confirm that the isolated RNA was free of DNA. Any other established primers could be used as well. Pure RNA cannot serve as a template in regular PCR amplification. Therefore the RNA template should not reveal any product, unless the sample still contains DNA residues. For further analysis, the RNA was reverse transcribed and the quality of the cDNA was confirmed again by β-actin PCR and gel electrophoresis. The established β-actin primers served as quality control in our laboratory, since they are designed to span an intron and therefore reveal products of different length for genomic DNA and RNA. If only one band appeared in the gel electrophoresis, it was concluded that the cDNA synthesis had been successful and the sample, free of genomic DNA, could be used for quantitative PCR.

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Figure 1.

Serial dilutions of cDNA amplified with Cyp1B1 primer are used to create a standard curve (here for example for Cyp1B1) with the LightCycler instrument.

To reveal gene expression and to compare the expression patterns between the smoker and the non-smoker, quantification was performed using real-time PCR with SYBR Green I detection. Optimal conditions for the target Cyp1B1 gene and the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been established (6).

The master mix, including all the PCR ingredients but the cDNA, was prepared in a separate room to prevent contamination. All the PCR contents needed such as PCR mix, primers, magnesium chloride stored at -20°C were thawed, vortexed and briefly centrifuged (1,000 rpm, 4 s) prior to use. It was very important to aliquot the PCR ingredients into small amounts to avoid repeated freeze-thawing cycles. The final amplification occurred in 1 x Absolute QPCR SYBR Green Capillary Mix® Fa. Abgene which contains an optimised reaction buffer and DNA Polymerase and is ideal for use with the Roche Light Cycler using glass capillaries, 600 nmol of the Cyp1B1 primer (MWG, Ebersberg, Germany) or 300 nmol of the GAPDH primer (Invitrogen, Karlsruhe, Germany) plus 2 μl cDNA in a total volume of 20 μl.

The sequence of the specific primers for CYP1B1 and GAPDH were CYP1B1 forward (5′-AACCGCAACTTCAGCAACTT-3′), CYP1B1 reverse (5′-GAGGATAAAGGCGTCCATCA-3′), and GAPDH forward (5′- TGCACCACCAACTGCTTAGC-3′), GAPDH reverse (5′-GGCATGGACTGTGGTCATGAG-3′).

To avoid aerosol contamination of the negative control while preparing the sample it is recommended to seal the negative control immediately before proceeding with the sample preparation. For Cyp1B1 amplification a touch down PCR program was used. Therefore the temperature (starting at 67°C) was reduced in three steps of two cycles, each time for 2°C before the amplification was carried out at 61°C (45 cycles). The amplification of the housekeeping gene GAPDH occurred at 61°C (45 cycles).

Since SYBR Green I detects double-strand DNA in general, melting curve analysis was used to check for primer dimers or unspecific PCR products. Serial dilutions of the cDNA (1:1; 1:10; 1:100; 1:1000; 1:10,000) with nuclease-free water were used to generate a standard curve (Figure 1) for Cyp1B1 as well as GAPDH which covered 3-5 orders of magnitude in the range of the samples in order to calculate the specific efficiency (E) using LightCycler Software 3.5. After completion of PCR, the LightCycler software calculates the copy number of target molecules by plotting logarithm of fluorescence versus cycle number and setting a baseline x-axis. The baseline identifies the cycle in which the log-linear signal can be distinguished from the background for each sample. The x-axis crossing point of each standard is measured and plotted against the logarithm of concentration to produce a standard curve. The concentrations of target sequence in the samples are extrapolated from the standard curve. The LightCycler Software 3.5. displays only the slope of a standard curve, which can be used to calculate the efficiency using the equation E=10^-1/slope.