cDNA synthesis and quantitative real-time PCR

Microarray data is inherently noisy and potentially full of artifacts, which can arise due to numerous experimental and technical reasons. Such artifacts include, for example, spurious signals caused by cross-hybridizing of single probes to se-quence-similar transcripts, artifacts induced by the sample preparation technique (e.g. preferential enrichment of certain transcripts during RNA amplification pro-cedures, degradation of RNA during the extraction process) and artifacts caused by the hybridization procedure itself. Additionally, large amounts of false-positives can potentially pass a statistical test, due to the high number of meas-urements initially introduced into the test. If, for example, all ~55,000 probe sets present on the HG U133 Plus 2.0 array are introduced into a statistical test and the p-value cutoff is chosen to be <0.05, which means that 5% of the probe sets pass the filter by chance, ~2,750 probe sets can be expected to pass the test as false-positives. Hence, a validation of microarray data by an independent method is indispensible.

Genes of interests extracted from the microarray data were validated by means of quantitative real-time PCR, with DNA complementary to mRNA of samples (cDNA) used as templates.

mRNA was transcribed to cDNA using SuperScript II reverse-transcriptase and oligo(dT) primers according to manufacturer’s instructions. For reverse-transcription of mRNA isolated from tissues, 1 µg total RNA was em-ployed as template. For reverse-transcription of mRNA extracted from cell, 2 µg total RNA were used. After synthesis, cDNA was diluted 1:10 with ddH2O, ali-quoted and stored at ‒20 °C. In parallel to each batch of cDNA synthesis, two control reactions were setup: One with ddH2O instead of RNA (H2O control) and a second one with ddH2O instead of reverse transcriptase (‒RT control). The H2O control was processed to check for contamination of the cDNA synthesis reagents,

whereas the ‒RT control was used to check for contaminations with genomic DNA within the RNA preparation.

Due to the exponential nature of polymerase chain reaction (PCR) amplifications, the quantitation of the amplicon after each cycle can be used to calculate the ini-tial amount of template. To do so, quantitative real-time PCRs using the TaqMan® system (Applied Biosystems, Life Technologies Corporation, Carlsbad, USA) were performed. Pre-designed “TaqMan® Gene Expression Assays”, which had been optimized by the manufacturer, were purchased for all genes of inter-est/target genes. Table 4 (page 41) summarizes the assays used. These “Gene Ex-pression Assays” contain target gene-specific DNA probe molecules 5’-linked to the fluorescent reporter dye 6-FAM^TM (6-carboxyfluorescein) and 3’-linked to a quencher – either BHQ-1 (black hole quencher 1) or MGB (minor groove binder).

Excitation of the reporter dye does not lead to fluorescence, as all emitted light is reabsorbed by the quencher in the reporter dye’s close vicinity. The probes hy-bridize specifically to exon-exon-boundaries of the template cDNA. A thermus aquaticus (Taq)-polymerase-based PCR-reaction using specific PCR primers, situated up and downstream of the probe, initiates amplification. During this proc-ess, the 5’ to 3’ exonuclease activity of the polymerase digests the probe, separat-ing reporter fluorophore and quencher. Thus, quencher molecules are not able to keep absorbing light emitted by the reporter. The resulting emission of light can be used to indirectly determine the amount of PCR product after each cycle by fluorescence measurement (Fig. 9). The “ABI PRISM® 7900 HT Sequence De-tection System”, which was used to measure the fluorescence after each cycle, subsequently plots the fluorescence intensity over the PCR cycles.

Figure 9: The principle of TaqMan®-PCR.

Figure adopted from Walter H. Koch (Koch 2004)

R – reporter fluorescent dye; Q – quencher; hυ – light quantum; Taq – thermus aquaticus polymerase

Additional to PCR for the target gene, each sample was also subjected to a PCR for an endogenous control (“housekeeping gene”) to allow quantitation of target gene expression, later on. Beta-actin (ACTB) was chosen as the endogenous con-trol for expression studies in gastric tumor samples. Its variation in expression across the investigated tumor samples was smaller than the one of the prominent

“housekeeper” glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a phe-nomenon that had been described previously elsewhere, too (Rubie et al. 2005).

The difference in inter-tissue expression variation is illustrated in Figure 10. For

expression analyses in cell lines, GAPDH remained to be the endogenous control of choice.

If expression of the target gene was lower than the expression of the endogenous control, PCRs for both genes were run in duplex in the same reaction. This simul-taneous detection is made possible by the availability of endogenous control ex-pression assays comprising probes labeled with a different fluorescent reporter dye (VIC®). If the expression of the target gene was higher than the one of the endogenous control, singleplex PCRs (with amplification of both genes being per-formed in individual reactions) were conducted. Later on, only results from either duplex or singleplex PCRs were compared to one another.

Figure 10: Inter-tissue expression variation of GAPDH and ACTB in human gastric adenocarcinomas.

Normalized (see chapter 2.2.11.1) expression values of probe sets covering the transcripts of GAPDH (217398_x_at, 212581_x_at, 213453_x_at) and ACTB (213867_x_at, 224594_x_at, 200801_x_at) were extracted from the microarray data set of all 59 samples. Means of respective expression values were calculated. Data distributions are displayed by box and whisker plots.

GAPDH ‒ glyceraldehyde-3-phosphate dehydrogenase; ACTB ‒ actin, beta

If available RNA amounts were “low”, TaqMan®-PCRs were performed using the “RNA UltraSense^TM One-Step Quantitative RT-PCR System”, which is espe-cially suitable for sensitive and reproducible detection of low-abundance RNA molecules.

Table 5 summarizes the different PCR preparation schemes used.

Table 5: Preparation of TaqMan®-PCR reactions.

conc. ‒ concentration

Primer-probe-mix for endogenous control 20× 0.5 1×

Sample (cDNA) 2.5

Primer-probe-mix for target gene or endogenous control 20× 0.5 1×

Sample (cDNA) 2.5

“UltraSense^TM One-Step”-based TaqMan®-PCR:

Multiplex reaction

Primer-probe-mix for endogenous control 20× 0.5 1×

ROX Reference Dye (internal reference) 0.2

Sample (RNA) 1.3

“Conventional” TaqMan®-PCR: “UltraSense^TM One-Step”-based TaqMan®-PCR:

Cycle: 50.0 °C 2 min Cycle: 50.0 °C 15 min

95.0 °C 10 min 95.0 °C 2 min

95.0 °C 15 s ← 95.0 °C 15 s ←

↑ ≥40× ↑ 45×

60.0 °C 1 min → 60.0 °C 30 s →

Quantitation of target gene expression was performed relative to the endogenous control using the comparative CT-method (∆CT-method), described in the “Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quanti-tative PCR” (Applied Biosystems, Life Technologies Corporation, Carlsbad, USA). In brief, the cycle, after which the signal erupts exponentially above a de-fined threshold, is called CT. The CT represents a logarithmic measure for the amount of template. To normalize the CT of the target gene, the difference to the CT of the endogenous control, the so-called delta CT (∆CT) is calculated. The anti-logarithmic value of the ∆CT is a value for the expression of the target gene rela-tive to the expression of the control. All TaqMan®-PCRs were performed in trip-licates. Arithmetic means of CT-values were calculated and standard error of the mean ∆CT was calculated by taking the square root of the sum of the squared standard errors of the mean of both individual CT values.