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RNA degradation patterns in cardiac tissues kept at different time intervals and temperatures before RNA sequencing.

Author(s): Jacobsen SB, Tfelt-Hansen J, Smerup MH, Morling N, Andersen JD

Publication: PLoS One, 2025, Vol. 20, Page e0323786

PubMed ID: 40373069 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA integrity and RNA sequencing metrics among nine case-matched cardiac tissue specimens stored for up to 28 days at 4°C or room temperature before RNA extraction. The right atrial appendage was collected from nine patients during cardiac surgery, and each was immediately divided into eighteen 3 mm³ pieces. Pieces were placed in Eppendorf tubes with 10 μl isotonic phosphate buffered saline (PBS) and stored in cardboard boxes for 0 (mean 45 min), 1, 7, 14, and 28 days at 4°C or 22°C (room temperature) (two pieces each). Tissue was homogenized for  2 min at 20 Hz using a TissueLyser II twice, and RNA was extracted using the RNeasy Fibrous Tissue Mini Kit. RNA integrity numbers (RIN) and DV200 were determined using a Bioanalyzer RNA 6000 Pico Assay Kit. RNA was quantified using a Qubit RNA HS Assay.  Sequencing libraries were prepared with the SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian Kit and sequenced using a NovaSeq 6000 instrument.

   Summary of Findings:

   RNA integrity numbers (RIN) and the percentage of fragments >200 nt (DV200) declined progressively with storage duration, with a more rapid decline observed when fresh cardiac specimens were stored at room temperature than when stored at 4°C. Storage duration and temperature did not affect the number of sequencing reads; and, gene expression levels were strongly correlated between replicate specimens (median ρ = 0.87), regardless of storage duration or temperature. The percentage of low-quality reads (Q<30), unmapped reads, reads aligning to intronic/intergenic regions and mitochondrial reads increased with the duration of cardiac specimen storage, with a larger increase observed with room temperature storage than storage at 4°C. In contrast, the percentage of protein-coding reads exhibited a larger decline with storage at room temperature than at 4°C. The percentage of reads that align to long non-coding genes (lncRNA) was not affected by the duration or temperature of cardiac tissue storage. When cardiac specimens were stored at 4°C, 15,448 (26.4%) genes had a slow decay rate (positive slope for the log-transformed expression levels at each time point) and 8,431 (14.4%) genes had a fast decay rate (negative slope for the log-transformed expression levels at each time point). When cardiac specimens were stored at 22°C, 4,462 (7.6%) genes had a slow decay rate and 12,648 (21.6%) genes had a fast decay rate. The decay rate was slow for ≥70% of mitochondrial transcripts, but up to 50% of the protein-coding transcripts had a fast decay rate. Genes classified as having a fast decay rate were longer than those classified as having a slow decay rate, but there was no clear effect of GC content on the decay rate. In principal component analysis of the gene expression data, principal component 1, which explained 36.1% of the variance, was weakly but significantly correlated with storage duration (R²=0.17, P<0.0001), storage temperature (R²=0.13, P<0.0001), RIN (R²=0.26, P<0.0001), DV200 (R²=0.20, P<0.0001), and patient age (R²=0.04, P<0.05) but was not significantly correlated with intra-individual differences. Similarly, principal component 2, which explained 12.36% of the variance, was weakly but significantly correlated with storage duration (R²=0.30, P<0.0001), storage temperature (R²=0.16, P<0.0001), RIN (R²=0.12, P<0.0001), and DV200 (R²=0.05, P<0.01) but was not significantly correlated with intra-individual differences. The number of genes found to differentially expressed after storage of cardiac specimens relative to 0 h controls increased with longer storage durations and higher temperatures.  Importantly, the effect was systematic, with most genes that were significantly different at one storage timepoint after 7 days were also found to be differentially expressed at another timepoint(s).  The genes that were differentially expressed included some cardiac genes. The authors found that when RIN or DV200 was used as a covariate, the number of differentially expressed genes among specimens stored at 4°C decreased, but inclusion of DV200 as a covariate only had a limited effect for specimens stored at 22°C.

   Biospecimens

   • Tissue - Heart

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Cardiovascular Disease

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Next generation sequencing
   RNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	4°C 22°C (Room temperature)
   Storage	Storage duration	0 days (mean 45 min) 1 day 7 days 14 days 28 days

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