Wilhelm Jochen, Muyal Jai Prakash, Best Johannes, Kwapiszewska Grazyna, Stein Maria Magdalena, Seeger Werner, Bohle Rainer Maria, Fink Ludger
Department of Pathology, Justus-Liebig-University Giessen, Giessen, Germany.
Clin Chem. 2006 Jun;52(6):1161-7. doi: 10.1373/clinchem.2005.062406. Epub 2006 Apr 20.
Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification.
We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases.
Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases.
Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.
从活检或激光显微切割获得的小生物样本通常无法产生足够的RNA用于成功的微阵列杂交；因此，在进行微阵列实验之前需进行RNA扩增。我们比较了两种常用的RNA扩增技术。
我们比较了两种市售方法，用于体外转录（IVT）的Arcturus RiboAmp和用于PCR的Clontech BD SMART，以预扩增从小鼠肝脏和肾脏分离的50 ng总RNA。通过实时PCR确定3个序列的扩增因子。使用10K 50聚体寡聚物点样微阵列（MWG Biotech）比较了技术内部和技术之间以及与未扩增样本的差异表达谱。通过与公共表达数据库比较，在转录本和蛋白质水平上验证了微阵列结果。
两轮IVT后特定序列的扩增因子低于12轮SMART后的扩增因子。此外，IVT显示随着扩增序列与polyA尾距离的增加，扩增明显减少，表明产生了较小的产物。在微阵列实验中，SMART后重复样本的重现性最高。此外，与未扩增样本以及表达数据库相比，经SMART处理的样本显示出更高的相关性。
每当一轮T7-IVT不能产生足够用于微阵列杂交的产物时（通常当以<200 ng总RNA作为起始材料时就是这种情况），我们建议使用SMART PCR进行预扩增。
