Formalin-fixed paraffin-embedded (FFPE) archival tissue samples serve as invaluable resources for disease research, particularly in the field of cancer. However, the processing of FFPE samples in molecular biology assays can be a formidable challenge due to the inherent degradation of nucleic acids caused by the fixation process and subsequent storage methods employed for tissue preservation. Consequently, RNA transcripts within FFPE samples are often fragmented, posing a hurdle to accurate analysis. Nevertheless, Illumina RNA enrichment assays offer a promising solution to address these challenges by directly capturing the coding regions of RNA, circumventing the need for poly(A) capture or amplification using site-specific primers. Accurate assessment of RNA quality is crucial to ensure reliable downstream analysis. In this article, we present a comprehensive guide to evaluating RNA quality from FFPE samples, offering insights into sample preparation, quality control measures, and practical recommendations for successful analysis.
Variability of FFPE Samples
FFPE samples exhibit inherent variability that can significantly impact the quality of RNA obtained. When evaluating FFPE samples, it is imperative to take into account the following factors:
- Fixation Time: Prolonged fixation times can exacerbate RNA degradation. Optimal fixation protocols should be tailored based on the specific tissue type and experimental requirements to minimize potential detrimental effects.
- Sample Age: Older FFPE samples tend to exhibit higher levels of RNA degradation. Consequently, the age of the samples should be carefully considered during the evaluation process to ensure accurate interpretation of results.
- Storage Conditions: Suboptimal storage conditions, such as elevated temperature or humidity, can accelerate RNA degradation within FFPE samples. A thorough assessment of the storage conditions, along with their potential impact on RNA quality, is crucial to account for variability and maintain data integrity.
Evaluating RNA Quality
Accurate assessment of RNA quality is paramount to obtaining reliable and robust RNA sequencing results. The following methods should be utilized for comprehensive evaluation:
- RNA Integrity Assessment: Determine RNA integrity using sophisticated techniques such as capillary electrophoresis (e.g., Agilent Bioanalyzer) or microfluidic platforms. These advanced methods generate informative metrics, including the RNA integrity number (RIN), which provides a quantitative measure of RNA degradation. Higher RIN values indicate superior RNA integrity.
- Fragment Analysis: Analyze the distribution of RNA fragment lengths using capillary electrophoresis or similar approaches. Longer fragment lengths are indicative of reduced RNA degradation and higher overall RNA quality.
- Total RNA Quantification: Accurately measure the concentration of total RNA utilizing precise spectrophotometric methods such as UV absorbance (e.g., Nanodrop) or fluorescent-based assays (e.g., Qubit). Reliable quantification enables estimation of the RNA content within FFPE samples, facilitating subsequent analysis.
- Reverse Transcription and PCR-based Quality Control: Perform reverse transcription of RNA samples, followed by PCR amplification of target genes. This step allows for the assessment of the success and efficiency of the reverse transcription and PCR processes, thereby indicating the functionality of RNA for downstream sequencing applications.
Optimization and Data Analysis
To maximize the efficacy of Illumina RNA enrichment assays, it is essential to optimize experimental parameters based on the evaluation of RNA quality. Fine-tuning sample input, hybridization conditions, and other relevant factors is crucial to ensure optimal outcomes. Additionally, it is advisable to employ robust bioinformatics tools and pipelines for meticulous data analysis, taking into account the inherent variability associated with FFPE samples. By adopting a comprehensive and systematic approach, researchers can overcome challenges inherent to FFPE samples and derive reliable and meaningful insights from their RNA sequencing endeavors.
