Template-switching anchored polymerase chain reaction reliably amplifies functional lambda light chain transcripts of malignant lymphoma

MT Koning, V Nteleah, H Veelken… - Leukemia & …, 2014 - Taylor & Francis
MT Koning, V Nteleah, H Veelken, MA Navarrete
Leukemia & lymphoma, 2014Taylor & Francis
There is increasing evidence of the role of the B-cell receptor (BCR) in non-Hodgkin
lymphoma pathogenesis, especially in diffuse large B-cell lymphoma, follicular lymphoma
and chronic lymphocytic leukemia [1–3]. Therefore, the accurate assessment of the BCR
sequence and structure is particularly important and requires the development of reliable
tools. The requirement of BCR signaling in the survival of mantle cell lymphoma (MCL) cells
[4], a highly restricted immunoglobulin (Ig) gene repertoire with stereotyped variable regions …
There is increasing evidence of the role of the B-cell receptor (BCR) in non-Hodgkin lymphoma pathogenesis, especially in diffuse large B-cell lymphoma, follicular lymphoma and chronic lymphocytic leukemia [1–3]. Therefore, the accurate assessment of the BCR sequence and structure is particularly important and requires the development of reliable tools. The requirement of BCR signaling in the survival of mantle cell lymphoma (MCL) cells [4], a highly restricted immunoglobulin (Ig) gene repertoire with stereotyped variable regions in the heavy and light chains [5, 6], and the promising results with novel therapies targeting the BCR [1, 7] indicate that the role of the BCR in lymphoma pathogenesis also extends to MCL pathogenesis, a lymphoid malignancy with a particularly poor prognosis.
Given the lack of in vivo experimental models and the difficult expansion of primary MCL cells ex vivo, MCL cell lines are frequently used as experimental models. However, essential information regarding their precise immunoglobulin sequences is incomplete. Recently, Pighi et al. made an important contribution by sequencing the rearranged heavy and light chain genes of several widely used MCL cell lines [6]. In this study, Ig genes were amplified from cDNA with a set of forward primers annealing to particular V or leader regions. It has been reported that this approach may introduce amplification bias or prevent the amplification of particular Ig rearrangements [6, 8, 9]. In fact, the authors report that in the case of the cell line Granta-519, the initial PCR reaction failed to amplify any product. With a second primer set an IGLV amplicon was obtained. However, this transcript was a truncated mRNA with a non-productive rearrangement. In order to overcome potential annealing issues arising from the use of forward primer sets, we have developed an anchored polymerase chain reaction (PCR) method in which Ig transcripts are amplified with an oligo-dC-containing “anchor” primer at the 5’end of the transcript and nested primers specific for the Ig constant region [3, 8]. This
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