Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

–Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18–24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3' barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3' barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

miRNAs are remarkably well conserved in formalin-fixed paraffin-embedded (FFPE) specimens^1,2,3. Previous work has demonstrated that the expression of these short regulatory non-coding single stranded RNA molecules can be successfully evaluated using total RNA from FFPE samples and provide relevant gene expression data when compared to the original fresh tissues^4,5,6,7,8. When compared to large-size messenger RNAs, ....

1. Preparation of All Reagents and Primers

1. Order all primers and adapters as described in Figure 1.
2. Prepare the calibrator cocktail stock, which will be spiked in each individual ligation.
   1. Resuspend the carrier oligonucleotide (Figure 1) to 0.5 µM with RNase-free water. Resuspend the 10 calibrator oligonucleotides (Figure 1) to 100 µM using the 0.5 µM carrier oligonucleotide solution.
   2. Combine 10 µL of each of the 10 calibrators in a siliconized microcentrifuge to obtain a 100 µM cocktail calibr....

As described in the method here, a total of 18 individual FFPE RNA samples (100 ng each) are set up in separate tubes to undergo 3' adenylated barcoded oligonucleotide T4 ligation overnight. The next day, the enzymatic reactions are heat-deactivated, combined, and precipitated in a single tube. The RNA pellet is resuspended and the ligated RNA molecules are separated on a 15% denaturing polyacrylamide gel (PAGE), where RNA oligonucleotide size markers that migrated in adjacent wells o.......

A highly reproducible and robust cDNA library preparation protocol for NGS of small RNAs archived in FFPE RNA specimens is presented in this protocol, which is a modified and optimized version of the procedure described by Hafner et al.²²

All steps of this protocol have been optimized for use with older archived and compromised total RNA recovered from FFPE specimens. The key step of this protocol, for processing small amounts of FFPE RNA, resides in the poolin.......

The authors wish to disclose that a publication containing some of the data presented in this manuscript was published in the International Journal of Molecular Sciences by Loudig et al.²¹.

We thank Dr. Thomas Tuschl, head of the laboratory for RNA molecular biology, as well as members of his laboratory for their support and for sharing the technology developed in his laboratory and providing access to the RNAworld pipeline. We also thank Dr. Markus Hafner for sharing his protocol and providing detailed descriptions on all biochemical and enzymatic steps used in his initial procedure.