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

Summary

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.

Abstract

–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.

Introduction

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, which have been shown to be critically affected by FFPE tissue processing (formaldehyde, heat, desiccation, etc.), endogenous RNases, and the age of the specimens, the small size of miRNAs (~18–24 nucleotides) appears to make them resistant to degradation and resilient to long-term storage, also demonstrated through miRNA expression studies that outperform high-throughput mRNA studies in archived specimens⁹. miRNA expression studies using archived clinical specimens, which have mostly been performed in small-scale analyses, have demonstrated that single or multiplexed quantitative PCR assays, different types of microarray technologies, and most recently NGS can be used to assess the expression of preserved miRNAs after optimization of these assays^{10,11,12,13,14}.

Given that dysregulation of miRNA expression has been associated with the development of a variety of human malignancies and that there is potentially an enormous supply of clinically annotated archived specimens, it has become apparent that these small RNA molecules represent a promising source of potential cancer biomarkers^15,16,17,18. The use of a high-throughput gene expression technology such as NGS has the advantage of providing a global evaluation of all miRNA transcripts when compared to targeted technologies such as PCR and/or microarrays¹⁹. For this reason, an optimized, affordable, and easily applicable protocol for cDNA library preparation of small RNAs from older archived specimens for NGS was optimized to enable large-scale retrospective studies²⁰.

We previously established a simultaneous RNA/DNA extraction protocol for separate recovery of RNA and DNA from older archived specimens, which we found to outperform contemporary commercial kits²¹. Using this extraction protocol, to obtain total RNA from FFPE tissues archived for extended period of times, we optimized the preparation of cDNA libraries for NGS of miRNAs preserved in clinical specimens for up to 35 years. Furthermore, in a recently published study where we prepared cDNA libraries from clinically classified ductal carcinoma in situ (DCIS) specimens, we identified differentially expressed miRNAs that were validated by quantitative PCR, which indicated that specific miRNA expression changes may be detectable in DCIS lesions from patients who develop breast cancer when compared to DCIS lesions from patients who do not develop breast cancer.

Considering the cost of commercial kits for preparation of small RNA cDNA libraries, the potential for their discontinuation, as well as the use of copyright/patent-protected reagents that cannot be optimized, we decided to adapt a previously published laboratory-based and kit-free 3' barcoded cDNA library preparation protocol for NGS of small RNAs archived in FFPE specimens, allowing simultaneous analysis of 18 samples²². This protocol provides an ideal and robust step-by-step procedure with visual and technical evaluation checkpoints, which were critical for adaptation to FFPE RNA specimens, and has a strong potential for application to other sources of compromised or difficult to use RNA material. The original protocol's applicability was improved by replacing radioactively labeled size markers with fluorescent (e.g., SYBR Gold) detectable RNA size markers used during selection of ligated libraries on large polyacrylamide gels. This optimized protocol relies on the ligation of 3' barcoded adapters to 18 individual FFPE RNA specimens, which are then pooled together to undergo 5' adapter ligation, reverse-transcription, and a pilot PCR analysis for tailored amplification of the final cDNA library prior to large-scale PCR amplification, purification, and NGS on a high throughput sequencer.

Protocol

1. Preparation of All Reagents and Primers Order all primers and adapters as described in Figure 1. Prepare the calibrator cocktail stock, which will be spiked in each individual ligation. 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 oligonuc…

Representative Results

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…

Discussion

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…

Materials

1% Triton x-100	Invitrogen	HFH10
10mM ATP	Ambion	AM8110G
10X dNTPs	Ambion	AM8110G
10x TBE	Thermofisher Scientific	15581044
14M Mercaptoethanol	Sigma	O3445I-100
20 nt ladder	Jena Bioscience	M-232S
20mg/ml Bovine Serum Albumine	Sigma	B8894-5ML
50X Titanium Taq	Clontech Laboratories	639208
Ammonium Persulfate	Fisher Scientific	7727-54-0
BRL Vertical Gel Electrophoresis System with glass plates and combs	GIBCO	V16
Dimethyl sulfoxide (DMSO)	Sigma	D9170-5VL
Eppendorf microcentrifuge 5424R	USA scientific	4054-4537Q
Eppndorf Thermomixer	USA scientific	4053-8223Q
Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tubes 1.5ml	Fisher Scientific	02-681-320
Gel Breaker Tube 0.5 ml	IST Engineering Inc,	3388-100
Gel electrophoresis apparatus 7cm x10cm- Mini-sub Cell GT with gel trays and combs	Biorad	1704446
Glycoblue	Ambion	AM9516
Jersey-Cote	LabScientific, Inc	1188
KcL 2M	Ambion	AM9640G
MgCl2 1M	Ambion	AM9530G
Minifuge dual rotor personal centrifuge	USA scientific	2641-0016
Model V16 polyacrylamide gel electrophoresis apparatus, glasses, combs, and spacers	Ciore Life Science	21070010
Oligonucleotides	IDT	Defined during order
Owl EasyCast B2 mini electrophoresis system- with gel trays and combs	Thermofisher Scientific	B2
Qiaquick Gel Extraction kit	Qiagen	28704
Restriction enzyme PmeI	NEB	R0560S
RNase-free water	Ambion	AM9932
Safe Imager 2.0	Life Technologies	G6600
Safe Imager 2.0 blue light transilluminator	Thermofisher	G6600
SeaKem LE agarose	Lonza	50002
Superscript III reverse transcription kit	Invitrogen	18080-044
SybrGold	Life Technologies	S11494
T4 RNA Ligase 1	NEB	M0204S
T4 RNA Ligase 2 Truncated K227Q	NEB	0351L
TEMED	Fisher Scientific	O3446I-100
Themocycler with heated lid	Applied Biosystem	4359659
Tris 1M pH 7.5	Invitrogen	15567027
Tris 1M pH8.0	Ambion	AM9855G
UltraPure Sequagel system concentrate, diluent, and buffer	National Diagnostics	EC-833