TransPlex

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TransPlex® RNA Amplification Kit

Ken Heuermann and Brian Ward
Research and Development, Biotechnology Division, Sigma-Aldrich

Abstract

The TransPlex Complete Whole Transcriptome Amplification (WTA2) Kit effectively amplifies intact and highly degraded RNA. To benchmark maintenance of representative RNA levels during amplification, differential gene expression of human liver and brain tissues was examined by microarray analysis. Expression profiles of high-quality RNA amplified with the TransPlex Complete WTA2 Kit or Eberwine linear amplification were compared with that of unamplified cDNA. Results indicate that the TransPlex Complete WTA2 Kit and unamplified profiles correspond closely. Effective amplification of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue was demonstrated by comparing the array results using target prepared from frozen or FFPE malignant prostate samples, versus matched frozen normal tissue. KEGG pathway profiles and global transcriptome analysis reveal that amplified FFPE target performed comparably to that of frozen malignant tissue. The TransPlex Complete WTA2 Kit is able to amplify nanogram quantities of intact total RNA or highly degraded RNA from FFPE tissue samples while maintaining transcript levels representative of that of the unamplified input RNA.

Introduction

The sequencing of the human genome has transformed etiological studies of human pathology and the pursuit of novel therapies. Until recently, validated drug targets, demonstrated to be associated with a particular disease or condition, were screened for their response to potential drug candidates. Target-based drug discovery has been increasingly replaced by phenotype-based approaches.1 Phenotype-based discovery relies on high-throughput and highcontent detection and screening processes to identify potential drug targets, which are de-convoluted by further experimentation and analysis. The gene expression microarray, for example, provides a high-content snapshot of sample-specific expression and is able to detect subtle, transcriptome-wide changes in mRNA levels resulting from disease, physiological development, or therapeutic treatment. These platforms have been successfully used to define disease markers.2,3,4,5 But whereas a relatively small number of RNA source samples may identify potential disease markers, validation requires a substantially larger sample pool. Inaccessibility to a sufficient number of fresh (or fresh frozen) clinical samples impedes the validation process, and consequently, progress towards development of novel treatments for disease.
Over the years, histological studies have amassed large repositories of well-characterized formalin-fixed paraffin-embedded (FFPE) tissue samples. Amplified genomic DNA from fixed tissues has yielded a significant body of relevant information.6 However, RNA lability and the effects of formaldehyde on RNA7 give rise to degradation and chemical modification. To date, the scope of application for FFPE RNA has been restricted to rt-qPCR,8,9,10 and a focused, diagnostic array platform.11,12 Efforts to utilize amplified FFPE RNA for whole transcriptome microarray applications have met with some success, however only when using mildly degraded RNA, still maintaining a distinct 28S/18S total RNA banding pattern.13
In this study, maintenance of relative transcript abundance during TransPlex Complete WTA2 Kit amplification is first validated with commercially prepared high-quality RNAs. Subsequently, microarray expression profiles generated with target prepared from highly degraded RNA extracted from FFPE tissue are shown to be comparable to those prepared from the matched frozen counterpart.

Materials

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Conclusions

Acknowledgements

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