Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

NOTE: Ethics statement: This protocol follows the guidelines of our institution, the Spedali Civili di Brescia

1. Preparation of a “Triple-Insert” Plasmid

1. Selection and preparation of appropriate starting material:
   1. Obtain a sample containing cells very likely to have TRECs and KRECs detectable by PCR, such as peripheral blood of a young healthy subject collected into EDTA tubes.
      NOTE: Originally, we had developed the method using thymocytes for TRECs and mononuclear cells from tonsil fragments for KRECs, but healthy subjects usually have an amount of TRECs and KRECs sufficient to allow their detection by PCR. Therefore, peripheral blood should be a valid alternative, easier to obtain and to work with. Considering that TRECs, in particular, decrease with age in healthy adults, peripheral blood from a young adult, if not from a children, is advised.
   2. Separate peripheral blood mononuclear cells (PBMC) by a standard density gradient separation method.
2. DNA extraction:
   1. Extract DNA from PBMC using a commercial DNA Blood Mini Kit. Follow the manufacturer’s instructions with the few modifications described below.
      1. Incubate the samples at 70 °C (instead of 56 °C) for 10 min using a shaker-incubator at 1,400 rpm.
      2. Add the elution buffer heated at 70 °C to the column, incubate for 5 min at 70 °C and elute DNA by centrifugation as per manufacturer’s instructions. Determine the extracted DNA concentration by spectrophotometric estimation at 260/280 nm.
3. Preparation of a plasmid containing the inserts of TREC and KREC signal joints (SJ) and TCRAC reference gene:
   1. Amplify the DNA extracted from PBMC (to obtain the TREC-SJ, KREC-SJ, and TCRAC fragments) with primers specific for TRECs, KRECs and TCRAC (see Table 1).
      NOTE: At the 5’-end, the KREC- and TCRAC-specific primers contain HindIII and SpeI restriction sites (in lower-case in the sequences shown Table 1) with an appropriate number of flanking additional nucleotides (indicated in italic); both features are not present in the canonical KREC and TCRAC sequences.

Table 1. Primers used for the cloning procedures. At the 5'-end, in lower case are shown nucleotides corresponding to restriction enzyme sites, whereas in italics are shown added flanking nucleotides.

3. 2. Perform PCR reactions in a final volume of 25 μl, consisting of 1x Buffer II, 1.5 mM MgCl₂, dNTP mix (200 µM each), primers at the final concentration of 900 nM, AmpliTaq DNA Polymerase (2.5 U/25 μl), and 100 ng of DNA.
   3. Use the following PCR parameters: first step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 sec; 60 °C for 30 sec; 72 °C for 30 sec,and finally 1 cycle of 72 °C for 10 min. PCR product lengths should be: 380 bp for TRECs, 166 bp for KRECs, 381 bp for TCRAC.
   4. Insert the PCR product of TREC-SJ in the T-A acceptor site of the pCR2.1-TOPO Vector. Transform the plasmid DNA in XL1-blue chemically competent cells by heat-shock following the protocol provided with the cells. Among the colonies that grow because of ampicillin resistance, identify those carrying the insert, which will appear white because of lost β-galactosidase complementation, and inoculate them into a master dish. Finally, identify the colonies containing the TREC fragment by PCR.
   5. Expand one of the identified colonies and purify the plasmid DNA using a plasmid miniprep kit and follow the manufacturer’s instructions. Digest the plasmid DNA and the TCRAC amplified product with SpeI restriction enzyme and ligate the TCRAC fragment into the SpeI restriction site.
   6. Transform the plasmid DNA in XL1-blue cells and identify the colonies containing the TCRAC fragment by PCR. Expand one of the identified colonies and purify the plasmid DNA using the plasmid miniprep kit.
   7. Digest the plasmid DNA and the KREC amplified product with HindIII and clone the KREC-SJ fragment into the HindIII restriction site. Transform the plasmid DNA in XL1-blue cells and identify the colonies containing the third fragment by PCR.
   8. Verify, by direct sequencing, the presence of the three inserts and the absence of mutations. The map of the final triple-insert plasmid is represented in Figure 1.

Figure 1. Triple-insert plasmid map. Triple-insert plasmid map showing the position of TREC, KREC, TCRAC sequences and restriction enzyme sites. Please click here to view a larger version of this figure.

3. 9. Expand one of the identified colonies, purify the plasmid DNA using a plasmid midiprep kit. Determine the plasmid DNA concentration by spectrophotometric estimation at 260/280 nm and the DNA quality by gel electrophoresis. Store appropriate aliquots of the plasmid at -80 °C (e.g., 50 μl each).

2. Standard Curve Preparation

NOTE: Prepare all dilutions in 0.1x TE buffer in a place specially designed for containment of DNA carry-over.

1. Calculate the mass of the plasmid copy numbers of interest (see Table 2), taking into account that the plasmid size is 4,846 bp and that the average mass per bp is 1.096 x 10^-21 g/bp, and therefore: m_p = (4,846 bp) x (1.096 x 10^-21 g/bp) = 5,311.216 x 10^-21 g = 5.311 x 10^-18 g.

Table 2. Mass of plasmid needed for each standard curve dilution point.

2. Calculate the concentrations of plasmid DNA by dividing the needed plasmid masses by the volume (5 μl) to be pipetted into each reaction (see Table 3).

Table 3. Calculation of plasmid concentrations needed for each dilution point.

3. Thaw an aliquot of the plasmid. Linearize 2 μg of the plasmid DNA with XhoI and determine its concentration by spectrophotometric estimation at 260/280 nm.
4. Prepare a proper dilution of the linearized plasmid DNA in order to achieve a convenient working stock solution to start from (e.g., 100 ng/μl = 0.1 μg/μl = 1 x 10^-7 g/μl). Store the remainder of the linearized plasmid DNA at -80 °C.
5. Perform a convenient number of 1:10 or 1:100 dilutions to bring the plasmid at the more workable concentration of 1 x 10^-10 g/μl.
6. Use the formula C₁V₁ = C₂V₂ to calculate the dilution volume (V₂ – V₁) needed to prepare the first standard curve point of the series (1 x 10⁶ copies, corresponding to 1.062 x 10^-12 g/5 μl; see Table 4).

Table 4. Dilution calculations.

7. Proceed with a regular series of 1:10 dilutions to obtain the remaining standard curve points.
   NOTE: Do not forget to vortex each plasmid dilution briefly before performing the next.
   NOTE: The highest dilution of the standard curve (10 copies/5 μl) must be prepared just before performing the assay, because such a small quantity of plasmid DNA is not stable enough to be stored for future use.
8. Store the triple-plasmid dilution points at -80 °C in separate tubes until use. The diluted triple-plasmid DNA is highly stable for at least 6 months.

3. DNA Extraction from Target Samples

1. Separate PBMC from peripheral blood collected into EDTA tubes using the density gradient separation method.
   NOTE: Alternatively, use other appropriate starting material (e.g., sorted lymphocyte subpopulations, whole blood) as target sample.
2. Extract the DNA from PBMC of target samples as reported in step 1.2.

4. Real-time PCR for Quantification of TRECs and KRECs

1. Plate preparation and loading:
   1. Arrange a PCR plate including at least two replicates for each samples to be analyzed: standard curve (A→F 1→4), positive control (CTRL⁺; G1→4) and no-template control (NTC; H 1→4).
      NOTE: Observe a typical scheme in Table 5, but different formats can be created. Remember that TRECs and KRECs will be amplified together in the same wells, distinct from those of TCRAC.

Table 5. Sample real-time PCR plate.

1. 2. Calculate the total number of wells needed (include 1 – 2 extra wells to account for possible pipetting errors) and prepare the master mix for TRECs/KRECs and TCRAC in separate tubes (see Table 6).
      NOTE: The primers and probes specific for TRECs, KRECs and TCRAC are mentioned in Table 7. The primer and probe final concentrations are 900 nM and 200 nM, respectively.

Table 6. Volume of reagents needed for the indicated wells.

Table 7. Sequence of primer and probes for the real-time PCR assay.

1. 3. Add 20 µl of each mix (TRECs/KRECs and TCRAC). Before leaving the reagent preparation room, seal the reaction plate with an optical adhesive cover.
   4. In a nucleic acid extraction room, lift the adhesive cover and add 5 µl of genomic DNA sample (400 – 500 ng) to the wells. Add 5 µl of genomic DNA (i.e., prepared from PBMC obtained from umbilical cord blood) to the CTRL⁺ wells.
      NOTE: As an alternative to umbilical cord blood, use DNA from the same known, positive peripheral blood sample or pool of samples, or prepare an “artificial” positive standard by mixing a determined amount of triple-insert plasmid with genomic DNA from TREC- and KREC-negative cell lines.
   5. Add 5 µl of water to the NTC wells. Seal the reaction plate again with the optical adhesive cover.
   6. Move to a place ensuring containment of plasmid DNA carry-over; thaw each dilution point of the plasmid DNA standard curve only before use and prepare the highest dilution (10 copies/5 µl). Lift up the adhesive cover only from A→H 1→4 wells and add 5 µl of each point dilution of plasmid DNA standard curve. Seal the adhesive cover again.
   7. Verify the absence of bubbles on the bottom of the wells, thus ensuring that the reagents are positioned at the bottom.
   8. Run the assay on a Real-time PCR system. The standard protocol consists of first step at 50 °C for 2 min, an initial heating at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 sec, and a combined primer/probe annealing and elongation at 60 °C for 1 min.
   9. Save the data at the end of PCR program.
      NOTE: To avoid DNA cross-contamination, remember: to follow the usual good laboratory practice recommendations for quantitative PCR when setting up the real time PCR assay: use separate areas/rooms for nucleic acid extraction, reagent preparation, plasmid manipulation, amplification reaction; use separate pipette sets; change gloves/coats as appropriate.
2. Results and calculation:
   NOTE: The following steps are based on our experience using the software provided with the real-time PCR instrument mentioned in the Materials Table, but, in general, they should apply to other real-time PCR analysis platforms and softwares. Keep in mind that, even if using the same software, usually there is more than one way to execute the required commands (e.g., icons, shortcuts).
   1. Open the saved result file in the software and click on the “Results” tab. On top of the window, click on “Analysis” menu and select “Analysis settings”. Let the software determine the threshold automatically by selecting “All” detectors and “Auto Ct” (Ct = threshold cycle. Let the software set “Automatic Baseline” for background elimination by default.
   2. Click on the tab named "Amplification Plot" and, at the rightmost part of the window, select one of the three "detectors" in the corresponding drop-down menu (e.g., start with TRECs). Just below, select "Manual Ct" to manually set a threshold; go to the lower part of the window and select the wells corresponding to the standard curve dilution points of the chosen detector to show the amplification plots. To do this just click and drag the mouse over the corresponding cells of the table.
      1. To adjust the threshold manually, drag the red threshold line up and down, and then click "Analyze" to reanalyze results. To check how setting the threshold affects the results, select the “Report” tab, and see the resulting Ct of the respective standard curve dilution points. When moving the threshold, try to comply with the following recommendations to get an optimal placement.
      2. Go back to the "Amplification Plot" tab and verify that the threshold is in a region of exponential amplification sufficiently above the background noise but below the plateau phase. If the amplification plot is not shown in logarithmic scale, right-click on the plot to recall the “Graph Settings” and set post-run Y-axis setting as "log", set the threshold half-way in the linear part of the plot, and observe the amplification curves approximately parallel to each other.
      3. Click on the “Report” tab to see the Ct results. Ensure that the threshold placement produced results that maximize the precision of the two replicates of each standard curve dilution points.
      4. Check that the threshold is placed at the point which best reflects the extreme orders of magnitude of the standard curve dilution points (optimal sensitivity). Repeat step 4.2.2 and remaining substeps for the remaining two detectors (KRECs and TCRAC).
      5. Go to the Report tab, drag the mouse again in the table at the lower portion of the window to select the cells corresponding to the wells of the standard curve for all detectors (TRECs, KRECs, TCRAC). Verify that the resulting Ct of the three targets is very similar at the same dilution points (e.g., difference not more than 0.5 Ct). Readjust thresholds if needed and re-check.
         NOTE: Because the triple-insert plasmid contains a single-copy of each target and therefore the amplification ratio should theoretically be close to 1:1:1. In some cases it may be necessary to manually adjust also the baseline for one or more of the detectors to get better results. Just recall the “Analysis settings” window, select the detector for which the adjustment is needed, then set “Manual Baseline”, and insert custom values for the Start and End Cycles (usually 3 to 15). Then click “OK & Reanalyze” and recheck previous points.
   3. Accept the experimental session only after verifying the following:
      1. Click on the “Report” tab, go to the lower portion of the window, select NTC wells and check that the corresponding Ct is “Undet” (i.e., no amplification is present in NTC wells).
      2. Select the “Standard Curve” tab and look to the right of the plot window; select detectors from the drop-down menu and see whether the reported standard curve slope ranges between -3.55 and -3.32 (corresponding to PCR efficiencies of 91 – 100%)
      3. Make sure that the coefficient of determination (R²) is higher than 0.998 (Figure 2), by reading its value right below the slope and intercept (in the same “Standard Curve” tab, after selecting each detector).
         NOTE: Pay attention to the extreme dilution points because their misalignment may affect the slope more easily (they have a high “leverage” effect on the regression line). In particular, keep in mind that the highest plasmid dilution may degrade sooner than expected. However, whenever possible it is useful not to discard them, because a rational approach may be to consider 100 as the limit of quantitative detection: in other words, for samples between 10 and 100, the result may be reported as <100 or “positive, but not quantifiable”, while if outside the curve range (<10) it may be reported as 0 or “undetectable”.
         NOTE: It may be useful to keep an archive of the Ct values of previous standard curve dilutions, in order to calculate an average value for each dilution point to be kept as a sort of optimal “reference” range or internal quality check for future experiments (e.g., calculate mean and SD to build Shewhart control charts). Analogously, it may be helpful to keep an archive of the past Ct of the positive control.

Figure 2. Standard curves for TRECs, KRECs, and TCRAC. Plots of the standard curve points and log-regression line estimates for TRECs (A), KRECs (B), and TCRAC (C) are built to verify compliance with the ideal exponential amplification rate (slope = 3.32) that would correspond to an efficiency of 100%. Ct: threshold cycle; R²: regression coefficient of determination. Please click here to view a larger version of this figure.

2. 3. 4. Click on the “Report” tab and verify that the Ct of the positive control (found in the corresponding cells of the table under the chart) is consistent with the results of the previous assays on the same known positive control.
         NOTE: The “Report” tab shows the amount of TRECs, KRECs and TCRAC of the samples being tested that the software has calculated by interpolation from the respective standard curve, using the Ct values obtained after the threshold and baseline have been properly set (a new analysis with a new threshold/baseline would change these results). To see where the interpolation takes place on the standard curve, select the “Standard Curve” tab, then select the desired wells, and look for the black “X” appearing on the regression line. Their y-axis value is the interpolated quantity. If using always the same positive control, appropriate reference ranges and/or quality control charts can be built, based on previous values determined on the positive control.
   4. Click on “File” menu, “Save”, and then “Export” to a .csv file to export the quantities of all wells in a text file containing comma separated values.
   5. Import the.csv file in a spreadsheet software and calculate the number of TRECs or KRECs per 10⁶ PBMC by setting the following formula appropriately: [(mean quantity of TRECs or KRECs) / (mean quantity of TCRAC/2)] x 10⁶
      NOTE: The mean quantity of TCRAC is divided by 2 because in each cell there are two TCRAC gene copies, e.g., one for each chromosome.
      NOTE: The consistency of the final number of TRECs and KRECs of positive control may be regarded, but keep in mind that values will not perfectly match because of the added variability of the standard curve dilution points. In case of gross divergence, the experiment may need to be repeated. Again, control charts may be built to set an appropriate reference range.
   6. (optional, but recommended) If the results of the complete blood count are available, calculate the TRECs or KRECs per ml of blood using the following formula:
      TRECs or KRECs per 1 x 10⁶) x (lymphocyte + monocyte count in 1 ml of blood) / 10⁶ = copies/ml.