Yexun Wang, Emi Arikawa, Min You, Shankar Sellappan, and Li Shen
QIAGEN, Fredrick, MD, USA
Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) samples can be challenging due to the process of tissue fixation, which causes chemical modifications that decrease the quality and availability of recovered RNA. This study demonstrates a novel preamplification step using gene-specific primers matched to a specific RT2 Profiler PCR Array, which results in an increase in the sensitivity of gene expression analysis of at least 100-fold.
Introduction
FFPE tissue samples represent an invaluable source of material for biomedical research and enable scientists to discover important links between molecular and clinical information. However, working with FFPE samples can be challenging due to nucleic acid crosslinking, fragmentation, and limited sample size. The ability to perform reliable gene expression analysis of FFPE tissue samples depends on the success of several steps in the workflow, including RNA extraction, reverse transcription (RT), qPCR primer design, and preamplification.
In this study, the results from a Human Cancer PathwayFinder RT
2 Profiler PCR Array show that introducing a preamplification step using gene specific primers that are matched to the RT
2 Profiler PCR Array increases the sensitivity at least 100-fold in real-time PCR gene analysis. With this RT
2 PreAMP technology, FFPE RNA can serve as a powerful tool for both retrospective and prospective gene profiling studies for biomarker identification and characterization.
Results and discussion
Considerations for successful gene expression analysis from FFPE samples
Careful consideration of every stage of the FFPE gene expression analysis workflow is necessary to achieve reliable results (see figure
Gene expression analysis of FFPE samples). RNA extraction from FFPE samples must ensure effective paraffin removal, de-crosslinking of RNA, RNA hydrolysis, and recovery of RNA fragments (for effective purification from of RNA from FFPE samples, we suggest the RNeasy FFPE Kit; click here for additional solutions for working with FFPE samples). Additional considerations include use of random or gene-specific primers for the RT reaction, designing qPCR primers for long or short amplicons, and strategies to increase signal sensitivity without altering the original gene expression profiles.
Choice of primers for efficient reverse transcription of FFPE samples
When working with FFPE RNA, fewer primers will generate cDNAs containing a target amplicon due to the high degree of RNA fragmentation that occurs during the FFPE fixation process (see figure
Reverse transcription of RNA from FFPE samples). The use of gene-specific primers for the RT reaction increases the probability that cDNA corresponding to the amplicon of interest will be produced (see figure
Gene-specific primers for RT from FFPE samples).
Considerations when designing primers for real-time PCR
PCR primers are often designed to generate amplicons that range in size from 130 to 200 bp. Although this is suitable for intact RNA, the high degree of FFPE RNA fragmentation means that smaller PCR amplicons are necessary to ensure amplification will occur (see figure
Effective primer design for qRT-PCR from FFPE samples).
Strategies for amplifying FFPE RNA without altering the gene expression profile
Preamplification of RNA samples can make it possible to perform more analyses on limited FFPE samples. However, it is essential that the method used amplifies all regions of the RNA equally to ensure that the gene expression profile of preamplified samples is equal to the gene expression profile of unamplified samples. RT
2 PreAmp Primer Mixes, which are designed specifically for RT
2 Profiler PCR Arrays, effectively amplify RNA without altering the gene expression profile (see figure
Highly comparable CT values from PreAMP and unamplified RNA isolated from an FFPE sample).
Gene expression analysis of RNA from FFPE samples using the Human Cancer PathwayFinder RT2 Profiler PCR Array
Due to small sample amounts, it is not always possible to obtain complete gene expression profiles using RNA from FFPE samples. Amplification of RNA using the PreAMP process enables quantification of genes that could not be detected without preamplification (see figure
The PreAMP process makes more genes quantifiable from FFPE samples).
When comparing the gene expression profiles of 2 different types of FFPE tissue (liver and intestine), comparable results were obtained whether the RT
2 PreAMP system or unamplified RNA was used (see figure
Comparable fold changes).
Conclusion
The RT
2 PreAMP system solves major challenges in real-time PCR-based gene expression analysis using FFPE samples. It greatly improves the lower limit of quantitation for FFPE RNA and also enables easy integration with the RT
2 Profiler PCR Array platform to analyze pathway-focused gene panels. With the improvement in both reverse transcription and qPCR primer design coupled with the introduction of a novel workflow tailored to the unique nature of these samples, better utilization of valuable FFPE sample resources is expected. These results demonstrate that PreAMP technology and the RT
2 Profiler PCR Array system is a valuable tool available for biomarker discovery or validation utilizing FFPE samples.
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