• Mouse B cell receptor (BCR)의 구성원인 IgG의 Heavy chain (H), Light chain (Kappa chain (κ), Lamda chain (λ))에 대한 NGS 서열 분석
• 하나의 샘플에서 Heavy chain (H), Kappa chain (κ), Lamda chain (λ)을 정밀하게 검출 및 분석
• Input sample: Spleen, lymph node, PBMCs, hybridoma에서 추출한 10ng - 3㎍ total RNA
• 높은 특이성과 감도로 BCR profiling

제품설명

B cell은 적응면역반응 (adaptive immune response)에서 필수적인 역할을 수행하며, B cell 표면에는 병원체의 특이적인 분자 패턴을 인지하는 B cell receptors (BCRs)를 발현하고 있다. BCR repertoire profiling (특이적인 H, K, L gene segments를 발현하는 receptor와 clonotype의 다양성을 검출)은 건강한 개체의 적응면역시스템에 대한 고찰뿐만 아니라, 여러 질환과 연관이 되어 있다. 그러므로 면역시스템에 의하여 발현되는 clonotype과 isotype을 정확하게 동정함으로써 B cell repertoire를 분석할 수 있다.
SMARTer® Mouse BCR IgG H/K/L Profiling Kit는 5’ RACE (Rapid Amplification cDNA Ends) 방법과 NGS 기술을 조합한 제품으로써 높은 특이성과 감도로 BCR profiling이 가능하다. 하나의 실험 반응에서 다수의 PCR 유전자를 증폭하는 기존의 Multiplex PCR을 이용한 방법은 특정 서열을 증폭함에 있어 감도, 특이성, 편향성(bias) 문제 등이 발생하는 것으로 알려져 있으며 이는 isotype 동정을 보다 까다롭게 만든다. 반면, 5’ RACE 방식은 BCR heavy chain 또는 light chain의 constant region 영역을 priming 하여 variable region을 증폭하므로, 보다 편향성이 적은 BCR profiling이 가능하다.


그림 1. BCR development.
The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.


그림 2. SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow.
Panel A. dT-primed first-strand cDNA synthesis is followed by two rounds of successive PCR for amplification of cDNA sequences. After post-PCR purification, size selection, and quality analysis, the library is ready for sequencing.
Panel B. Two rounds of successive PCR for the amplification of cDNA sequences corresponding to variable regions of BCR IgG heavy chain or BCR light chain (kappa or lamda) transcripts.


그림 3. PCR pooling affects alignment and on-target percentages.
Libraries containing BCR heavy and light chain sequences were generated using the SMART Mouse BCR kit as described in the user manual. If only heavy and kappa light chain PCR products are pooled and sequenced ("HK" in the table), a high percentage of the sequences align to IG reference sequences (using MiXCR software version 1.8) for each sample. If the lambda light chain PCR products are also pooled ("HKL" in the table), alignment percentages drop. However, the majority heavy chain and kappa chain sequences (top two clones) are identical and in the same proportion for each sequencing sample. CDR3 amino acid sequence, which defines the affinity of the antibody, is given for each clonotype shown.


그림 4. Accurate amplification of all five subclasses of IgG.
Libraries containing BCR heavy and light chain (kappa) sequences were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from an in-house hybridoma (10E8) and two ATCC hybridoma samples (HB-8117 and TIB-127) with different IgG subtypes. Panel A. Bioanalyzer traces show gene-specific amplification of heavy or light chains of each hybridoma. In each sample, the expected peak between 700-900 bp is observed. The strong discrete peaks in the electropherograms show that the kappa chain is being expressed in these hybridomas. In contrast, the lambda chain sequencing library is comparatively broad and of a smaller size range than expected. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics determined using MiXCR software (version 1.8), aligned against all IG sequences. Output from the MiXCR software for the top two clones shows the V, D, and J alleles and the isotype and subclass as identified by the software.

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