This method can answer key questions about NK cell receptor genetics such as haplotype diversity and how this relates to disease. Visual demonstration of this method will help to set the protocol in your laboratory, including the automation process and data analysis. The main advantages of this technique are that, unlike conventional methods, qKAT, which stands for quantitative KIR automated typing, is high-throughput and it provides gene copy number.
qKAT can provide insight into disease association. The implications of this technique can be useful for therapy for viral infections like HIV and hepatitis C, in cancer, in stem cell transplantation, and pregnancy disorders. qKAT can also be applied in studying population genetics as well as exploring expression and functional interactions between KIR and HLA molecules.
qKAT consists of 10 multiplex reactions that specifically target exons of the two KIR genes and one reference gene. In this protocol, specific primers are designed to detect the alleles of the given gene and PCR results in short amplicons. After this, dual-labeled specific hydrolysis probes are used to monitor the PCR amplification in real time.
Copy number analysis uses relative quantification of the target KIR genes to a reference gene. qKAT is designed and set up to be a high-throughput technique. Hence, each reaction can be performed on just under 1, 000 samples.
The following steps show the steps involved in the preparation and plating out of each 96-well DNA plate. To begin, after quantifying DNA, dilute the DNA in a 96-well deep-well plate. Include at least one control sample with a known copy number and one non-template control.
Centrifuge the plate at 450 gs for two minutes. Then, dispense each sample in quadruplicate into 384-well qPCR plates. After this, air dry the DNA in a clean area at room temperature for 24 hours.
To set up one qKAT reaction for 10 384-well plates, first, defrost the qPCR buffer, primer, and probe aliquots at four degrees Celsius. Prepare the master mix on ice according to the text protocol. Then, use a multi-channel pipette to distribute the master mix evenly across a 96-well deep-well plate.
Dispense 9.5 microliters of the master mix into each well of the plate with the dried gDNA samples. After this, seal the plate with foil and immediately store it at four degrees Celsius. Remember to perform a short water wash to clean the needles of the liquid handling system if dispensing master mix on more than one 384-well DNA plate.
Centrifuge the plate with the gDNA samples at 450 gs for three minutes. Then, incubate the plate at 4 degrees Celsius to resuspend the DNA and dissipate any air bubbles. After incubating the plate overnight, repeat the centrifugation to dissipate any air bubbles.
Then, place the plate in the cooled storage dock. Connect the qPCR machine to a microplate handler. Then, program the microplate handler according to the text protocol.
Once the run is complete, have the robot collect the plate from the qPCR machine and place it in the discard dock. After completing the amplification, open the qPCR software. Under the Navigator tab, open the saved reaction experiment file for a plate.
Next, open the Create New Analysis window and select the appropriate settings for the 2nd Derivative Max method. Then select Filter Comb and ensure the data collected for STAT6 is selected. Select the appropriate color compensation.
Click Calculate, and then click save file. To analyze the data with the Fit Points method, open the Create new analysis window and select the appropriate settings for the Fit Points method. Then, select the correct filters and color compensations for STAT6.
Set the noise band to exclude the background noise. After this, open the Analysis tab and set the fit points to three, and select Show Fit Points. Save changes and repeat the steps with appropriate settings for Fit Point and 2nd Derivative analysis for the KIR genes.
Open the Navigator tab in the qPCR software and select Results Batch Export. Then, open the folder containing the experiment files and transfer them to the right side of the window. Click Next, and select the name and location of the export file.
After this, select the analysis type of interest and click Next. Ensure the name of the file, the export folder, and the analysis type are correct before clicking Next to start the export process. When the export status changes to OK, the screen will automatically move to the next step.
Ensure that all of the selected files have been exported successfully and click Done. Then use the scripts in the text protocol to split the exported plates into individual reactions and to convert the files into a software readable by copy number analysis software. Next, open the copy number analysis software and select Import real-time PCR results file to load the text files created by the scripts.
Finally, select Analyze, and conduct the analysis by selecting Most frequent sample copy number. In this protocol, semi-automated typing, or qKAT, was used to copy number type KIR genes. The most frequent copy number for KIR2DL4 was two copies while the most frequent copy number for KIR3DS1 was zero.
While attempting this procedure, it is extremely important to make sure that the DNA is quantified accurately, to conduct all the steps on ice, and to make sure that the reagents are covered from light. It is also important to perform thorough checks on the raw data for samples that do not conform to the known LD rules for KIR genes. Following this procedure, other methods like sequencing can be performed in order to answer additional questions, for example, presence of fusion genes, or identification of novel alleles.
After its development, this method paved the way for researchers in the field of immunogenetics to explore the role of KIR in NK cell biology, disease resistance, and human reproduction.
