Martinelli G, Testoni N, Montefusco V, Amabile M, Saglio G, Ottaviani E, Terragna C, Bonifazzi F, de Vivo A, Pane F, Rosti G, Tura S
Institute of Haematology & Medical Oncology, Seràgnoli University of Bologna, Italy.
Haematologica. 1998 Jul;83(7):593-601.
Capillary electrophoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis).
The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 microgram of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per microgram of total RNA examined).
CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two types of bcr-abl junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (SD) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/microgram total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value 128,859 vs. 331,722 in patients with 0% blast cells and > 1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score < 0.8 and > 1.2, respectively; p = 0.003).
Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.
毛细管电泳（CE）已成为替代平板凝胶分析的一种有吸引力的方法，可直接、准确地检测扩增产物，几个循环的聚合酶链反应（PCR）就足以进行定量和定性分析。我们试图评估：1）CE是否可作为一种实用的非同位素方法，用于直接检测通过逆转录（RT）-PCR获得的扩增bcr-abl的存在（定性分析）；2）是否有可能使用竞争性RT-PCR并通过测量CE电泳图的峰面积来定量PCR产物（定量分析）。
从34例慢性髓性白血病（CML）患者诊断时的骨髓样本中提取1微克总RNA，通过RT-PCR产生两种与CML相关的bcr-abl转录产物（定性分析）（中位年龄47.5岁；范围18 - 65岁；中位索卡尔评分0.9；范围0.53 - 2.78）。PCR产物通过2%琼脂糖凝胶上的平板凝胶电泳（SGE）和CE进行分析（共128次运行；每个样本中位分析3.3次）。此外，我们通过竞争性RT-PCR方法和CE评估PCR产物的量（定量分析）（bcr-abl转录本以每微克检测的总RNA中的转录本表示）。
定性RT-PCR获得的PCR产物的CE分离显示，对应于两种bcr-abl连接类型的两个峰实现了基线分离：b2-a2型（343个碱基对，10例患者）在9.33分钟时显示出来[标准差（SD）=0.1]，b3-a2型（418个碱基对，24例患者）在10.03分钟时显示出来（SD = 0.25）。通过定量分析，我们发现在诊断时bcr-abl表达存在很大的患者间差异：bcr-abl转录本量的中位值为78,0０0个bcr-abl转录本/微克总RNA，范围为17,300至750,0０0。诊断时bcr-abl转录本的量与原始细胞数量有关（原始细胞为0%和>1%的患者中，平均值分别为128,859和331,722；p = 0.004）以及索卡尔评分有关（索卡尔评分<0.8和>1.2的患者中，平均值分别为156,865和408,800；p = 0.003）。
我们的结果证实，在该白血病研究中，CE分析在检测和定量bcr-abl PCR产物方面具有更高的分辨率和灵敏度。通过CE对bcr-abl产物进行定性分析为分析亚纳克量的DNA片段提供了一种快速技术（不到20分钟）。CE运行时间短，毛细管可重复使用，通过计算机控制步骤进行数据采集实现完全自动化可能是可行的。通过CE对bcr-abl进行竞争性/定量分析可减少反应次数并实现更精确的定量。
