Polymerase chain reaction (PCR)-based methods

PCR is a powerful tool that enables amplification and subsequent detection and quantification of DNA and RNA targets (Figure 14). The PCR reaction mix ideally consists of DNA/cDNA template, and a master mix that contains the polymerase enzyme, necessary nucleotides, forward (Fwd) and reverse (Rev) specific primers in final optimized concentrations.

Occasionally a fluorescent labeled probe is used. The PCR reaction volume is usually 50, 25, or 10 µl.

Figure 14. Illustration of main steps of PCR

The amplification of DNA occurs in cycles (35-40). Each PCR cycle consists of several steps that vary in duration and temperature according to the specific application. In general, three main steps are involved: 1-Denaturation step: usually at 95 ºC for 15-30 sec. The aim of denaturation is to separate the double stranded DNA into single strands. 2-Annealing step: during this step, the forward and reverse primers specifically bind (anneal) to their

complementary nucleotide sequences in the DNA template. The annealing temperature depends on the melting temperature of each primers. Common commercially available

primers are usually optimized to anneal at 60 ºC for 1 min. 3-Elongation (extension) step: usually at 72 ºC for 1 min. In some cases, the annealing and extension steps are combined in one step.

3.2.8.2 Cytokine gene expression

Cytokine genes were quantified using a two-step reverse transcriptase PCR (RT-PCR) method (163). In the first step, RNA was extracted from sorted γδ T cells (TCR γ/δ T cell isolation kit, Miltenyi) using the PureLink™ RNA Mini Kit (Invitrogen, ThermoFisher) and converted to cDNA using Super- Script™ IV VILO™ Master Mix (ThermoFisher) according to manufacturer’s instructions. The cDNA can be used immediately or kept at -20°C.

In the second step, cDNA products resulting from the 1st step were used as template for amplification of specific gene segments using predesigned TaqMan Gene Expression Assays for IL-1B, IL-2, IL-6, IL-7, IL-8, IL-12B, IL-15, and IL-17 genes and human ACTB gene as reference gene (Thermofisher scientific). PCR was performed using a 7500 fast real-time PCR instrument (Applied Biosystems).

3.2.8.3 TREC and KREC assessment

Somatic recombination occurs in all TCR genes. This TCR rearrangement process results in production of circular DNA products known as T cell receptor recombination excision circle (TREC). However, not all TRECs are appropriate for laboratory assessment.

A good candidate TREC must be detectable in peripheral blood and not affected by the different rearrangement possibilities. As rearrangements of TRB and TRG loci are very early events, their resulting TRECs are extensively diluted before they enter the peripheral blood rendering their levels in the PB below the detection limit (88).

Alternatively, somatic recombination of TRA locus demands the deletion of the TRD gene locus as it is interspersed with TRA along the same chromosomal location. Since this deletion occurs at a relatively late developmental stage, the generated TRECs are less subjected to the dilution effect of thymocyte expansion. Furthermore, this rearrangement results in production of δRec-ΨJα signal joint and coding joint in 70 % of cases (Figure 15) (164).

Likewise, B cells undergo somatic recombination of their immunoglobulin heavy and light chains. The rearrangement process of the kappa gene results in kappa deleting recombination excision circle (KREC) that can be used as surrogate for newly produced naïve B cells (165). In the first paper, we used a TaqMan real-time PCR based technique to quantify TREC and KREC levels from genomic DNA extracted from CD3+ and CD19+ sorted samples,

respectively (Figure 16) (165). Briefly, a multiplex PCR reaction was developed for

TREC/GAPDH in a final volume of 25 µl, consisting of 12.5 µl of TaqMan™ 2X Universal PCR Master Mix (Applied Biosystems), sjTREC primers (forward 5'- CAC ATC CCT TTC AAC CAT GCT; reverse 5'-GCC AGC TGC AGG GTT TAGG) and probe (6-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT) were added in a final concentration of 300 and 200 nM respectively, and GAPDH primers (forward 5'-GGA CTG AGG CTC CCA CCT TT; reverse 5'-GCA TGG ACT GTG GTC TGC AA) and probe (VIC-CAT CCA AGA CTG GCT CCT CCC TGC) were added in a final concentration of 150 nM and 100 nM

respectively. For KREC quantification, we used KREC primers and probes that were described by Sottini et al., (165)in a final concentration of 900 nM and 200 nM respectively and GAPDH as for TREC. PCR amplification was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR cycling conditions were as follows: 1× (95°C for 10 min), 40× (95°C for 15 sec, 60°C for 1 min). TREC and KREC values were calculated relative to GAPDH using the formula 2(∆Ct), where ∆Ct = Ct GAPDH- Ct TREC (KREC)

Figure 16. Illustration of the TaqMan PCR method used for TREC and KREC quantification

3.2.8.4 Telomer length (TL) assessment

TL was assessed by quantification of telomere DNA relative to the DNA of a single copy gene (T/S) as described before (166). Briefly, for each sample, telomere and human Beta globulin (HBG) gene DNA were amplified separately as triplicate in a MicroAmp Fast Optical 96 well plates, using SYBR green PCR technique.

A reaction mix was prepared separately for telomere and HBG with a total reaction volume of 20 µl containing Power SYBR green PCR Master Mix (Applied Biosystems) in a final concentration of 1×. For telomere DNA, forward and reverse primers in a final concentration of 100 and 900 nM, respectively, were added. Whereas for HBG DNA, forward and reverse primers in final concentration of 300 and 700 nM, respectively, were added. DNA samples were finally added to each well in a final amount of 25 ng. The PCR was performed on a 7500 fast real-time PCR-system (Applied Biosystems) using the following PCR conditions: 1× (10 min 95°C), 40× (15 sec 95°C, 1 min 60°C). Mean Ct values were determined for both telomere and HBG. And T/S ratio was calculated as follow: T/S = 2∆Ct where ∆Ct = (mean Ct HBG – mean Ct telomere). The following primers were used: Tel1b-Forward: CGG TTT GTT TGG GTT TGG GTT TGG GTTTGG GTT TGG GTT; Tel2b-Reverse: GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT; Hbg1-Forward: GCT TCT GAC ACA ACT GTG TTC ACT AGC; Hbg2-Reverse: CAC CAA CTT CAT CCA CGT TCA CC

Importantly, SYBR green based method can result in non-specific amplifications. Therefore, results should be carefully analyzed to avoid false results. In order to minimize this

limitation, the following measures were implemented: 1- Appropriate controls were used; non template control and positive control sample obtained from acute myeloid leukemia cell line (THP-1). 2-Melting curve analysis was performed after amplification (Figure 17) to discriminate non-specific amplifications. 3-All samples were run as triplicates. Furthermore, samples from different time points for the same recipient were included in the same run simultaneously, to minimize plate to plate variations

Figure 17. Melting curve performed following SYBR green PCR for TL. To the right a melting curve generated from specific amplification. To the left a melting curve from non-specific amplification

3.2.9 TCR repertoire analysis