Checking primers for single amplicon specificity

To test the performance and validity of IL-6 and GAPDH primers, a preliminary primer check was performed. Using 1 µl (~100-200 ng) of PBMCs cDNA as template and 2 X SensiMixTM SYBR Green NO ROX kit (BioLine, UK), 20 µl real- time PCR reactions (one for each IL-6 primer set and one for GAPDH with its negative contol) were prepared and run as described in Chapter 2. The PCRs were run in two independent experiments and at the end of the cycling programme the melting curves of the PCR reactions were checked. Ideally, melting curve should contain a single peak with no shoulders, and the agarose gels of the amplified product should reveal a single band corresponding to the predicted amplicon length. Figure 4.2 shows the melting curves and agarose gel electrophoresis for the IL-6 primers used for the optimisation steps. Apart from the pre-optimized primer from Qiagen, all other primer sets gave rise to melting curves with more than one peak and more than one band on agarose gel indicating either the production of primer dimers or nonspecific products. The primer dimers may be eliminated by optimizing primer concentration or changing thermocycling parameters. Many attempts were made to optimize primer concentrations and cyclic parameters but all these attempts failed. Since these were very time consuming procedures, all primers with more than one peak during melting curve analysis were excluded and the pre-optimized primer set (Qiagen) was selected for the final experiments. Regarding the specificity of the GAPDH primer set the melting curve clearly indicated a single peak and demonstrated that the GAPDH PCR product had a specific melting temperature at ~82 °C (Figure 4.3 A & B). Subsequently, the agarose gel electrophoresis showed that the band was specific with the expected length of 133 bp (Figure 4.3 C).

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Figure 4.2: IL-6 primers test. Four IL-6 primer sets (selected from published papers) and one pre-optimized IL-6 primer set (purchased from Qiagen) were tested using 1µl (~100-200 ng) of PBMC cDNA and 2 X SensiMixTM SYBR Green NO ROX kit. A) Real-time fluorescent detection of the IL-6 amplicon. B) Dissociation (melting) curves of the different IL-6 PCR reactions. A single peak suggested the absence of non-specific amplification products. C) Agarose gel electrophoresis of real-time PCR products. Lane *: 100-10000 bp DNA Ladder; lane 1: IL-6 primer 1 (expected product size is 80 b.p); lane 2: IL-6 primer 2 (expected product size is 99 b.p); lane 3: IL-6 primer 3 (expected product size is 143 b.p); lane 4: IL-6 primer 4 (expected product size is 193 b.p); lane 5: commercial pre-optimized IL-6 primer (expected product size is 107 b.p). Apart from the pre-optimized IL-6 primer set, all other primers gave rise to non specific PCR products. These primers were excluded from the final study.

2 3 4 5

1

*

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C

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Figure 4.3: Quality control of the GAPDH primer set. The GAPDH primer set was tested using 1 µl (~100-200 ng) of PBMC cDNA and the 2 X SensiMixTM SYBR Green NO ROX kit. Real-time PCR was run in 20 μl volumes with 1 μl of forward and 1 μl of reverse primers. PCR conditions were as follows: 95°C for 10 min, 40 cycles of [95°C for 15 seconds, 60°C for 15 seconds and 72°C for 20 seconds]. The melting of the PCR product was performed from 55°C to 95°C, rising in 0.5°C increments. A) Real-time fluorescent detection of GAPDH amplicon. B) Melting curve for GAPDH PCR reaction. The single peak suggests a single size product. C) Agarose gel electrophoresis of the GAPDH real-time PCR products. The left lane shows the 100- 10000 bp molecular weight ladder. Lane 1 shows the amplicon (133 bp). No amplification was found in negative control (no template DNA sample, lane 2).

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155 4.3.2 Testing the primers amplification efficiency

The typical method to analyze real-time PCR data is the Livak method (Livak and Schmittgen, 2001). This is also referred to as the 2-ΔΔCT method. It uses only Ct values and consists of three main steps:

1) Normalization of the Ct value of gene of interest to that of reference gene, for both the test sample and the control sample. This is ΔCT:

ΔCt (test) = Ct (Gene of interest, test) – Ct (reference gene, test)

ΔCt (control) = Ct (Gene of interest, control) – Ct (reference gene, control)

2) Calculation of the difference between the ΔCT of the test sample and the ΔCT of the control. This is called ΔΔCt:

ΔΔCt = ΔCt (test) – ΔCt (control)

3) Calculation of the expression ratio:

Expression ratio (folds) = 2–ΔΔCT

However, this method often assumes that both the gene of interest and the reference gene PCR assays have 100% amplification efficiency (a two-fold increase in the amount of the amplicon during each cycle). Since 100% efficiency is rarely seen in practice, therefore the error rate in the fold difference increases exponentially. PCR amplification efficiencies between 90-110% are generally considered acceptable and enable PCR assays to accurately analyze gene expression using the Livak method (Dorak, 2011). To determine the PCR amplification efficiency a calibration standard curve is required. The cDNA template of known concentration is serially diluted. After the end of the cycling, the dilution and Ct values are exported to Microsoft Excel and a standard curve is drawn by plotting the Ct values versus the log10 dilution values

156 (linear regression) and the slope is calculated from the standard curve. The efficiency of the reaction can be calculated by the following equation: E = 10 (-1/slope) –1. Slopes of -3.1 to -3.6 in the Ct Vs. log-template amount standard curve correspond to a PCR efficiency of 90-110%. The closer the slope is to -3.33, the closer the amplification efficiency is to the theoretical 100% (Dorak, 2011). If the PCR efficiency is < 90%, the quantitative real-time PCR should be further optimized or alternative primers designed. If the efficiency is > 110%, running another standard curve experiment with a minimum of 3 replicates and a minimum of 5 logs of template concentration is recommended. Nowadays most of real-time PCR machines are provided with software that can generate the PCR efficiency calculations rapidly and automatically.

The preliminary primer test experiment has indicated all the IL-6 primers gave rise to non specific bands apart from the pre-optimized primers from Qiagen. Since these primers are pre-optimized by the company for the IL-6, therefore no PCR amplification efficiency test was carried out on these primers. However, the GAPDH reference gene primers were evaluated for their amplification efficiency. Here 4 serial 10 fold dilutions of PBMCs cDNA were made. Each dilution was prepared in triplicates and the PCR was run using the same parameters as in the previous experiment. As can be seen from Figure 4.4, the amplification efficiency of the GAPDH PCR reaction was 99%, R=0.99064, R2 = 0.98137. Therefore, these primers were used for the final experiments.

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Figure 4.4 Amplification efficiency of the GAPDH primer set using SensiMixTM SYBRGreen NO ROX kit. A) Real-time amplification curve from a 10-fold dilution series of a GAPDH target amplified from PBMCs cDNA with starting amounts of 100 ng. GAPDH was amplified in four triplicate reactions using the GAPDH experimental primer set. Real-time PCR was run in 20 μl of final volume with 1 μl of forward and 1 μl of reverse primers. The PCR conditions were as follows: 95°C for 10 min, 40 cycles of [95°C for 15 seconds, 60°C for 15 seconds and 72°C for 20 seconds]. B) Melting curve analysis. Single peaks on the melting curve of all samples indicated that no contaminating DNA, primer dimers or non-specific products were present in the reaction. C) Log-transformation of the fluorescence measurements and the points of intersection of the amplification curves and the threshold. The threshold was determined by the Rotor Gene 5 software. D) Standard curve of the GAPDH amplification. The curve generated by the Rotor Gene 5 software provided with the Rotor Gene 2000 Thermocycler based on the log dilution (X-axis) and Ct values (Y- axis). The efficiency of the PCR reaction was 99%. The correlation coefficient of the GAPDH standard was R = 0.99.

A B

158 4.3.3 Involvement of different NK cell activating receptors in IL-6 mRNA expression and protein secretion

4.3.3.1 Stimulation via CD16

1. Engagement of CD 16 induced IL-6 mRNA expression in PBNK cells