The overall goal of the following experiment is to estimate thymic and bone marrow output by quantifying both T-cell receptor excision circles or REX and K deleting recombination excision circles or corrects in a single assay. This is achieved by first constructing a triple insert plasmid containing fragments of the DNA sequence of TRX corrects and of a single copy reference gene in order to maintain a one to one to one ratio between the three targets after DNA amplification. Next, a standard curve is built by performing serial dilutions of the triple insert plasmid, which allows the absolute quantification of the three target sequences by real realtime PCR.
Finally, a realtime PCR assay is run leading to the absolute quantification of tres and cracks. The reported results show the changes of thymic and bone marrow output with age and gender in healthy children and adults. We first had the idea for this method when we needed to analyze both the bone marrow and thymic output in newborns or infants, considering that the amount of blood usually drawn is scarce compared to adults.
Therefore, we decided to combine the two methods of tracks and corrects quantification into a single assay in order to optimize sample preparation and reagent costs. The implication of this technique extend toward the diagnosis of primary and acquired immune deficiencies, but the method can also be used in all diseases involving loss of TMB cells as well as to study new reconstitution following bone marrow transplantation Through this method can provide insight into human disease. It can also be applied to basic research principally that involving lymphocyte homeostasis.
The main advantage of is the by employ a triple surplus mid only a single standard cure is needed, thus reduce the experimental variability. Generally, individuals new to this method may struggle because of the lengthy procedure required for the triple insert plasmid preparation or because of the issues related to standard curve preparation. To begin obtain a sample containing cells very likely to have TRX and cracks detectable by PCR, such as peripheral blood of a young healthy subject collected into EDTA tubes using a standard density gradient separation method.
Separate peripheral blood mononuclear cells after extracting the DNA from the cells according to the text protocol, use primers specific Fort TRX REX and TCR alpha constant gene to amplify Rex signal, joint Rex signal joint and TCR alpha constant gene fragments. Insert the PCR product of Trax signal joint in the TA acceptor site of the PCR 2.1 topo vector and use the plasmid DNA to transform XL one blue chemically competent cells by heat shock isolate ampicillin resistant white colonies that have lost beta lactase complementation onto a new master dish and use PCR to identify the colonies containing the trek fragment. After growing one of the trek positive colonies and isolating the DNA, use SP one to digest the plasmid and the TCR alpha constant gene amplified product and ligate the TCR alpha constant gene fragment into the plasmid.
Repeat transformation, colony selection and expansion and DNA isolation to insert the correct fragment, use hind three rather than SP one to digest the plasmid and the correct PCR fragment and repeat the process to ligate and isolate the DNA. Once the sequences of the three inserts have been verified, expand the colony and purify the plasmid DNA by MIDI prep according to the text protocol to prepare a standard curve. After calculating the mass and concentration of the plasmid as shown here, thaw an aliquot of the plasmid and you show one to linearize two micrograms with the linearized aliquot of DNA.
Use the formula C one V one equals C two V two. To calculate the dilution volume needed to prepare the first standard curve point of the series as shown here, prepare a series of one to 10 dilutions to obtain the remaining standard curve points. Store the plasmid dilution at negative 80 degrees Celsius until use To prepare samples for realtime PCR arrange a PCR plate that includes at least two replicates for each sample to be analyzed in the following manner with two separate tubes, one for Rex and cracks, and another one for TCR alpha constant.
Prepare the master mixes for the total number of wells needed. Add 20 microliters to the wells of the plate. Next, add five microliters of genomic DNA extracted from peripheral blood mononuclear cells or P BMCs.
Add five microliters of water to the no template control wells before using an optical adhesive cover to seal the reaction plate. Then thaw the plasmid DNA standard curve dilution and prepare the highest dilution. Uncover only the wells located between rows A through H and columns one through four.
And after brief vortexing, add five microliters of each dilution point to its designated. Well ensure that there are no bubbles at the bottom of the wells and that the reagents are settled at the bottom. Use a real-time PCR system to run the assay with the following protocol.
50 degrees Celsius for two minutes and initial heating at 95 degrees Celsius for 10 minutes, followed by 45 cycles of denaturation at 95 degrees Celsius for 15 seconds. And a combined primer probe e kneeling and elongation at 60 degrees Celsius for one minute. To analyze the real time PCR data, open the saved result file in the software and click on the results tab on top of the window.
Click on the analysis menu and select analysis settings. Let the software determine the threshold automatically by selecting all detectors and auto CT for threshold cycle. Let the software set automatic baseline for background elimination by default.
Click on the tab named amplification plot, and at the right most part of the window, select one of the three detectors in the corresponding dropdown menu. Just below select manual CT to manually set a threshold at the lower part of the window, select the wells corresponding to the standard curve dilution points of the chosen detector to show the amplification plots by clicking and dragging the mouse over the corresponding cells of the table. 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 to get optimal placement in the amplification plot tab, 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 the post run Y ais setting as log.
Set the threshold halfway in the linear part of the plot and observe the amplification curves approximately parallel to each other. 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 curves.
Dilution points. Check that the threshold is placed at the point which best reflects the extreme orders of magnitude of the standard curve dilution points under 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, verify that the resulting CT of the three targets is very similar at the same dilution points.
For example, difference, not more than 0.5 CT readjust thresholds if needed, and recheck under the report tab at the lower portion of the window. Select NTC wells and check that no amplification is present in the NTC wells, which corresponds to a CT of undetermined. Select the standard curve tab and to the right of the plot window, select detectors from the dropdown menu and see whether the reported standard curve slope ranges between negative 3.55 and negative 3.32, which corresponds to PCR efficiencies of 91 to 100%Ensure that the coefficient of determination or R squared is higher than 0.998 by checking its value just below the slope and intercept.
Click on the report tab and in the corresponding cells of the table under the chart, verify that the CT of the positive control is consistent with the results of the previous assays on the same known positive control. 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. Finally, import the CSV file in a spreadsheet software and calculate the number of tracks or corrects per 10 to the six PBMs by setting the following formula appropriately.
The assay described in this video was performed on a representative sample of 87 Healthy Controls. 42 children aged zero through 17 and 45 adults 24 through 60. As shown here, the number of treks decreased sharply with age due to thymic involution from zero to three to four years in adults.
The trek number also depends on gender because it decreases more rapidly in men than in women, which can impact the ability to set appropriate reference ranges. These graphs show that the number of cracks, however, decreased with age similar to the pattern seen with T TREXs only in children. Whereas in adults, the bone marrow output is fairly stable throughout life and does not depend on gender.
The triple in insert plasmid preparation requires about two or three weeks, but thereafter, the stored plasmid allots will last three years. The remaining part of the technique can be done in only four to five hours if performed properly. While attempting this procedure, it's very important to perform all plasmid related manipulation in a dedicated area to avoid the DNA cross-contamination.
It's also essential to verify the stability of the standard tube when analyzing results After its development. This technique provided researchers in the field of clinical immunology or hematology, an improved tool for the identification of primary immune deficiencies to monitor the outcome of immune reconstitution, following bone marrow transplantation, or to simply study lymphocyte homeostasis. After watching this video, you should have a good understanding of how to obtain the triple in plasmid, prepare the standard curve, run the realtime PCR experiment and perform the necessary calculations needed to obtain a reproducible absolute quantification of trax and cracks.
