Home › Immunology › qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes
Immunology JoVE (Open Access) Citable · DOI

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

DOI: 10.3791/58646-v
What you'll learn
  • ✓ Perform semi-automated KIR gene copy number typing using qPCR
  • ✓ Prepare DNA samples and master mixes for high-throughput KIR assays
  • ✓ Analyze qPCR data and calculate KIR gene copy numbers from results
  • ✓ Apply qKAT method to population and disease association studies
Protocol

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

Difficulty
intermediate
Total time
~3-4 hours per batch (includes qPCR run time of ~2 hours)
Biosafety
BSL-1

Steps

1
Prepare and plate out DNA samples

Prepare DNA samples and distribute them into qPCR plates for analysis. Ensure proper sample handling and plate organization for downstream processing.

▶ 01:45
2
Prepare master mix for qPCR reactions

Combine all reagents including primers, probes, and polymerase into a master mix. Prepare sufficient volume for all samples plus controls.

▶ 02:25
3
Run quantitative PCR assay

Load master mix into prepared DNA plates and execute qPCR protocol on thermal cycler. Monitor amplification and fluorescence data collection.

▶ 03:36
4
Perform post-run analysis of qPCR data

Analyze raw qPCR output including Ct values and fluorescence curves. Quality-check data for accuracy and consistency across samples.

▶ 04:07
5
Export results and calculate copy numbers

Export analyzed data and apply copy number calculation algorithms to determine KIR gene type for each sample.

▶ 05:19
6
Interpret qKAT results for KIR typing

Review representative qKAT outputs demonstrating successful copy number typing of KIR genes for population studies.

▶ 06:39
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