Department of Clinical Genetics, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht, The Netherlands.
Mol Biol Rep. 2012 Jul;39(7):7429-33. doi: 10.1007/s11033-012-1575-2. Epub 2012 Feb 17.
A rapid and easy method to screen for aberrant cDNA would be a very useful diagnostic tool in genetics since a fraction of the DNA variants found affect RNA splicing. The currently used RT-PCR methods require new primer combinations to study each variant that might affect splicing. Since MLPA is routinely used to detect large genomic deletions and successfully detected exon skipping events in Duchenne muscular dystrophy in cDNA, we performed a pilot study to evaluate its value for BRCA1 cDNA. The effect of puromycin, DNase I and two different DNA cleaning protocols were tested in the RNA analysis of lymphocyte cultures. We used two samples from unrelated families with two different BRCA1 exon deletion events, two healthy unrelated controls and six samples from hereditary breast/ovarian cancer syndrome (HBOC) patients without BRCA1/2 mutations. Using RNA treated with DNase I and cleaned in a column system from puromycin-treated fractions, we were able to identify the two BRCA1 deletions. Additional HBOC patients did not show additional splice events. However, we were not able to get reproducible results. Therefore, the cDNA-MLPA technique using kit BRCA1 P002 is in our hands currently not reliable enough for routine RNA analysis and needs further optimization.
一种快速简便的筛选异常 cDNA 的方法将成为遗传学中非常有用的诊断工具，因为发现的部分 DNA 变异会影响 RNA 剪接。目前使用的 RT-PCR 方法需要新的引物组合来研究可能影响剪接的每个变体。由于 MLPA 通常用于检测大片段基因组缺失，并成功地在 Duchenne 肌营养不良症的 cDNA 中检测到外显子跳跃事件，我们进行了一项初步研究来评估其对 BRCA1 cDNA 的价值。在淋巴细胞培养物的 RNA 分析中，我们测试了嘌呤霉素、DNase I 和两种不同 DNA 清洁方案的效果。我们使用来自两个具有不同 BRCA1 外显子缺失事件的无关家族的两个样本、两个健康的无关对照和六个来自遗传性乳腺癌/卵巢癌综合征 (HBOC) 患者的样本，这些患者没有 BRCA1/2 突变。使用经 DNase I 处理并用柱系统从嘌呤霉素处理部分清洁的 RNA，我们能够鉴定出这两个 BRCA1 缺失。其他 HBOC 患者没有显示出其他剪接事件。然而，我们无法获得可重复的结果。因此，目前我们手中使用试剂盒 BRCA1 P002 的 cDNA-MLPA 技术对于常规 RNA 分析来说还不够可靠，需要进一步优化。
