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3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage.

ABSTRACT: This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3' massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

SUBMITTER: Boneva S

PROVIDER: S-EPMC7498368 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage.

Boneva Stefaniya S Schlecht Anja A Böhringer Daniel D Mittelviefhaus Hans H Reinhard Thomas T Agostini Hansjürgen H Auw-Haedrich Claudia C Schlunck Günther G Wolf Julian J Lange Clemens C

Laboratory investigation; a journal of technical methods and pathology 20200528 10

This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3' massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixat ...[more]

PMID: 32467590

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Project description:IntroductionWe have recently reported about a novel technique for the generation of bioartificial vascular grafts based on the use of a compacted fibrin matrix. In this study, we evaluated the effects of a dehydration process on the biomechanical properties of compacted fibrin tubes and whether it allows for their long-term storage.Materials and methodsFibrin was precipitated from fresh frozen plasma by means of cryoprecipitation and simultaneously with a thrombin solution applied in a high-speed rotating casting mold. Subsequent dehydration of the fibrin tubes (29/38) was performed in dry air with a dilator inside the tube to prevent the collapse of the lumen. Dehydrated fibrin tubes were stored for six (n=9) and 12 months (n=10) at room temperature. Comparative analysis was done on initially generated and dehydrated fibrin tubes before and after storage to evaluate the effects of the dehydration process and storage on the biomechanical properties and structure of the tubes.ResultsThirty-eight fibrin tubes were generated by high-speed rotation-molding from 142±3 mg fibrinogen with an inner diameter of 5.8±0.1 mm and a length of 100 mm. A centrifugal force of nearly 900×g compacted applied fibrin, while fluid was pressed out of the matrix and drained from the mold via holes resulting in a 16-fold compaction of the fibrin matrix. Dehydration was characterized by shrinkage of the tubes to a diameter of 3.2±0.2 mm, while the length remained at 100 mm equivalent to a further two-fold compaction. The biomechanical strength of the dehydrated fibrin tubes significantly increased to values comparable to that of native ovine carotid arteries and maintained during the first 6 months of storage. After 12 months of storage, only five of 10 tubes were intact, and only one showed maintained biomechanical strength.DiscussionCompaction of a fibrin matrix in high-speed rotation-moulding and subsequent dehydration enables for the construction of small-caliber fibrin grafts. Over and above, the dehydration process allows their storage and stockpiling as a prerequisite for clinical use.

Project description:Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.

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3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage.

Publications

3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage.

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