KIR genes were genotyped for the presence or absence of the 17 KIR genes, including KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 3DL1, 3DL2, 3DL3, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1 (in the full-length form), 1D (in the deleted form) and two pseudogenes, 3DP1(putative protein product) and 2DP1(no protein expression) using PCR-SSP method with a commercially available KIR GENOTYPING SSP KIT (Invitrogen, California, USA). The PCR amplification was performed with the PCR system in a reaction mixture volume of 10 μl consisting of 4 μl pre-aliquoted PCR buffer, 0.06 μl Taq polymerase, and 30 to 50 ng of template DNA. Temperature cycling conditions for PCR reactions were as follows: denaturation for 1 minute at 95 °C, followed by 30 cycles for 20 seconds at 94 °C, 20 seconds at 63 °C, 1.5 minutes at 72 °C, a elongation step for 10 minutes at 72 °C and finally hold in 4 °C. PCR products were visualized under ultraviolet light after electrophoresis in 1.5% agarose gel well mixed with 10% v/v ethidium bromide. Negative controls were performed for each gene while positive internal controls were performed for each lane. False reactions that yielded no internal control bands were repeated.
Kir genotyping ssp kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The KIR GENOTYPING SSP KIT is a laboratory equipment product used for the detection and identification of killer cell immunoglobulin-like receptor (KIR) genes. The kit employs the sequence-specific primer (SSP) method to determine the presence or absence of specific KIR genes in a sample.
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5 protocols using kir genotyping ssp kit
1
Comprehensive KIR Genotyping by PCR-SSP
2
Killer Immunoglobulin-like Receptor Genotyping
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Killer immunoglobulin-like receptors genotyping was performed on DNA extracted from 400 μl of blood using QIAmp Blood Mini kit (Qiagen) with the KIR Genotyping SSP kit (Invitrogen) according to manufacturer’s instructions. The AA haplotype was defined as KIR2DL5Aneg/KIR2DL5Bneg/KIR2DS1neg/KIR2DS2neg/KIR2DS3neg/KIR2DS5neg/KIR2DS1neg.
3
KIR Genotyping for HSCT Donors
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Peripheral blood samples from donorerecipient pairs undergoing HLA-identical sibling HSCT were evaluated for KIR genes. Genomic DNA was extracted from peripheral blood mononuclear cells using the Promega DNA Extraction Kit (Promega, Madison, WI) following the manufacturer's instructions. Genotyping for 17 KIR genes was performed by PCR with sequence-specific primers using the KIR Genotyping SSP Kit (Invitrogen, Carlsbad, CA) to test for gene presence. A common combination of 9 genes (KIR3DL3, 2DL3, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4, and 3DL2) is often defined as the "A" haplotype. The group A haplotype encodes mainly for inhibitory receptors (KIR2DL3, KIR2DL1, and KIR3DL1) and appears to carry KIR2DS4 as its only stimulatory gene. KIR3DL3, KIR3DL2, KIR3DP1, and KIR2DL4 are the framework genes. KIR 2DS4*0010101-00104 (FUL) encode for cell surface receptors while the remaining full-length KIR2DS4 protein: KIR2DS4 has eight alleles currently described: only 2DS4*001-002 encodes for a cell surface receptor while the remaining alleles, 2DS4*003,*004, *006, *007, *008, carry a deletion in the exon encoding the membrane-proximal extracellular domain altering their reading frame with loss of the transmembrane and cytoplasmic domains have been described KIR1D.
4
KIR Gene and KIR-HLA Typing Protocol
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The presence or absence of KIR genes was determined using a commercial kit based on a sequence-specific primer amplification method – SSP (SSP KIR Genotyping kit – Invitrogen, Brown Deer, Wisconsin, USA). A total of 14 KIR genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, and 3DS1) and 2 KIR pseudogenes (2DP1 and 3DP1) were screened using this approach. KIR group genotype nomenclature was designated according to the current working definition, which characterizes genotypes AA and Bx based on the combinations of haplotype A (absence of all the activating genes, except KIR2DS4) and haplotype B (presence of one or more of the activating genes) [12 (link), 56 (link)]. We also classified KIR genotypes by the ID assigned by the Allele Frequency Net Database (http://www.allelefrequencies.net ). KIR-HLA pairs were named according to the presence of at least one allele of a particular allotype (Bw4 or Bw6; C1 or C2), as follows: Bw4/Bw4, Bw4/Bw6, Bw6/Bw6 and C1/C1, C1/C2, C2/C2.
5
KIR Genotyping Protocol using SSP
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Qualitative analysis of KIR genes was performed using a commercial kit based on sequence-specific primer amplification methods -SSP (SSP KIR Genotyping Kit, Invitrogen, Brown Deer, Wisconsin, USA). A total of 14 KIR genes and 2 KIR pseudogenes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1) were screened using this approach. KIR AA and Bx genotypes designation followed the current working definition, which characterizes these genotypes based on the combinations of haplotype A (absence of all the activating genes, except KIR2DS4) and haplotype B (presence of one or more of the activating genes) (Uhrberg et al., 1997; (link)Fernandes-Cardoso et al., 2016) (link).
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