Trenkle T, Welsh J, Jung B, Mathieu-Daude F, McClelland M
Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121, USA.
Nucleic Acids Res. 1998 Sep 1;26(17):3883-91. doi: 10.1093/nar/26.17.3883.
A method is presented in which the reduced complexity and non-stoichiometric amplification intrinsic to RNA arbitrarily primed PCR fingerprinting (RAP-PCR) is used to advantage to generate probes for differential screening of cDNA arrays. RAP-PCR fingerprints were converted to probes for human cDNA clones arrayed as Escherichia coli colonies on nylon membranes. Each array contained 18 432 cDNA clones from the IMAGE consortium. Hybridization to approximately 1000 cDNA clones was detected using each RAP-PCR probe. Different RAP-PCR fingerprints gave hybridization patterns having very little overlap (<3%) with each other or with hybridization patterns from total cDNA probes. Consequently, repeated application of RAP-PCR probes allows a greater fraction of the message population to be screened on this type of array than can be achieved with a radiolabeled total cDNA probe. This method was applied to RNA from HaCaT keratinocytes treated with epidermal growth factor. Two RAP-PCR probes detected hybridization to 2000 clones, from which 22 candidate differentially expressed genes were observed. Differential expression was tested for 15 of these clones using RT-PCR and 13 were confirmed. The use of this cDNA array to analyze RAP-PCR fingerprints allowed for an increase in detection of 10-20-fold over the conventional denaturing polyacrylamide gel approach to RAP-PCR or differential display. Throughput is vastly improved by the reduction in cloning and sequencing afforded by the use of arrays. Also, repeated cloning and sequencing of the same gene or of genes already known to be regulated in the system of interest is minimized. The procedure we describe uses inexpensive arrays of plasmid clones spotted as E.coli colonies to detect differential expression, but these reduced complexity probes should also prove useful on arrays of PCR-amplified fragments and on oligonucleotide chips. Genesobserved in this manuscript: H11520, U35048, R48633, H28735, M13918, H12999, H05639, X79781, M31627, H23972, AB000712, R75916, U66894, AF067817.
本文介绍了一种方法，该方法利用RNA任意引物PCR指纹图谱（RAP-PCR）固有的降低复杂性和非化学计量扩增的优势，来生成用于cDNA阵列差异筛选的探针。RAP-PCR指纹图谱被转化为用于筛选尼龙膜上以大肠杆菌菌落形式排列的人类cDNA克隆的探针。每个阵列包含来自IMAGE联合体的18432个cDNA克隆。使用每个RAP-PCR探针可检测到与大约1000个cDNA克隆的杂交。不同的RAP-PCR指纹图谱产生的杂交模式彼此之间或与总cDNA探针的杂交模式几乎没有重叠（<3%）。因此，与放射性标记的总cDNA探针相比，重复应用RAP-PCR探针能够在这种类型的阵列上筛选出更大比例的信息群体。该方法应用于用表皮生长因子处理的HaCaT角质形成细胞的RNA。两个RAP-PCR探针检测到与2000个克隆的杂交，从中观察到22个候选差异表达基因。使用RT-PCR对其中15个克隆进行了差异表达测试，13个得到证实。使用这种cDNA阵列分析RAP-PCR指纹图谱比传统的变性聚丙烯酰胺凝胶法进行RAP-PCR或差异显示检测能力提高了10到20倍。通过使用阵列减少克隆和测序，通量得到极大提高。此外，同一基因或已知在感兴趣系统中受调控的基因的重复克隆和测序也被最小化。我们描述的方法使用作为大肠杆菌菌落点样的廉价质粒克隆阵列来检测差异表达，但这些降低复杂性的探针在PCR扩增片段阵列和寡核苷酸芯片上也应证明是有用的。本文中观察到的基因：H11520、U35048、R48633、H28735、M13918、H12999、H05639、X79781、M31627、H23972、AB000712、R75916、U66894、AF067817。
