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Quality assessment of RNA in long-term storage: The All Our Families biorepository.

Author(s): Stephenson NL, Hornaday KK, Doktorchik CTA, Lyon AW, Tough SC, Slater DM

Publication: PLoS One, 2020, Vol. 15, Page e0242404

PubMed ID: 33259520 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA purity, yield, and integrity in RNA extracted from PAXgene blood stored for an average of 12 or 100 months before RNA extraction. The effects of trimester of collection, outdoor temperature on the day of collection, which phlebotomist performed the collection, time at -20˚C, and which of the three freezers was used for storage on the association between storage duration and RNA purity, yield, and integrity was also evaluated. Blood was collected into four PAXgene tubes by one of five different phlebotomists from each of 165 pregnant women during the second trimester and from 161 of the women again in the third trimester. Blood was stored temporarily at -20°C for an average of 55 days before storage of aliquots in one of three different freezers at -80°C for an average of 12.34 months (2011 extraction) or 99.64 months (2018 extraction) until RNA extraction. RNA was isolated from the blood specimens in 2011 (all 165 second trimester specimens and 161 third trimester specimens) and 2018 (141 second trimester and 137 third trimester specimens) using the PAXgene Blood RNA Kit. RNA yield and purity were evaluated by spectrophotometer and integrity was assessed by bioanalyzer.

   Summary of Findings:

   The mean RNA purity (260/280 ratio) was lower in RNA extracted in 2011 than 2018 (2.07 versus 2.10, P<0.0001). The authors constructed a linear regression model showing a mean increase of 0.009897 per month of storage with no effect of trimester of collection, outdoor temperature on the day of collection, which phlebotomist performed the collection, time at -20˚C, or which of the three freezers was used for storage. While the RNA yields were statistically comparable between extractions performed in 2011 (mean: 13.08 µg, range: 1.32-39.16 µg) and 2018 (mean: 12.64 µg, range: 1.32-39.16 µg). However, linear regression revealed a 0.90 µg loss per month of storage that was unaffected by trimester of collection, outdoor temperature on the day of collection, which phlebotomist performed the collection, time at -20˚C, or which of the three freezers was used for storage. In contrast to RNA yield and purity, no effect of storage duration on RIN was observed (mean 8.49, range 7.2-9.5), but there was a 0.03 increase in RIN per month of storage after adjusting for which phlebotomist performed the collection and outdoor temperature on the day of collection. Importantly, the authors attribute the increased purity and RIN with storage to better precision during the extraction and analysis at the second timepoint. Further, the authors state that while significant, none of the changes observed would make a meaningful difference on downstream analysis.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • PAXgene

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	Mean 12.34 months Mean 99.64 months Range of 5-130 months Difference of 80-93 months

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