Short-Term Stability Study of RNA at Room Temperature

Author(s): Mathay C, Yan W, Chuaqui RF, Skubitz A, Jeon JP, Fall N, Betsou F, Barnes M

Publication: Biopreservation and Biobanking, 2012, Vol. 10, Page 531-542

Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storing RNA dried with or without stabilizers on the stability of RNA from blood specimens. Blood was collected into PAXgene tubes and stored for 2 h prior to automated RNA extraction. RNA was stored at -80 degrees C until shipment at -80 degrees C to one of 6 centers. At the centers, RNA was dehydrated in one of 3 matrices and stored for 2 weeks before shipment to one of three testing laboratories where samples were rehydrated with water. Additional RNA samples were mailed directly to the testing laboratories and stored in liquid form at -80 degrees C or room temperature for an unspecified duration. RNAse was added by contact with human skin for 3 sec.

   Summary of Findings:

   RNA recovery was comparable between samples that were mailed frozen or at room temperature in the liquid state, or samples that were dried and stored for 2 weeks in RNAstable, GenTegra or RNAshell, but only 81% as much RNA was recovered from dried samples stored for 2 weeks without the presence of a matrix. Samples that had been stored dried in GenTegra had a lower mean 260/280 ratio than the other specimens. RINs were not significantly affected by drying samples, type of matrix or storage temperature, but increased variability in RIN was found when liquid or dried RNA was stored without a matrix. Compared to liquid samples that were mailed and stored at -80 degrees C, cycle threshold (CT) values were higher for samples stored dried in GenTegra for 2 weeks, regardless of testing facility (3 facilities) or gene target. A 1.55 cycle increase in beta actin CT was found using RNA stored in liquid form at room temperature by 1 of 3 facilities, but all other samples performed similarly regardless of storage condition or facility. CT values for RNA stored dried in GenTegra could be decreased by using less RNA, but further purification of RNA with PicoPure eliminated this inhibitory effect. Analytical variability in beta-actin was lowest when RNA samples were stored dried for 2 weeks without matrix, followed by those stored dried in RNAstable, and then those stored as liquid at -80 degrees C. After 3.5 months of storage at room temperature, only samples stored as liquid RNA showed degradation, but the effects of storing RNA dried in GenTegra were not assessed. Further, testing showed that RINs of RNA stored dried in RNAstable were comparable after 12 months to those of samples stored at -80 degrees C. RNAse contamination led to decreased RIN values and increased CT values, regardless of matrix type or storage condition, but the decrease in RIN values was smaller for RNAse contaminated specimens stored in RNAshell than for samples with other storage matrixes.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Between site transportation method	Mailed
   Storage	Specimen transport duration/condition	-80 degrees C Room temperature
   Storage	Time at room temperature	2 weeks 3.5 months 12 months
   Biospecimen Preservation	RNA stabilization method	Freezing GenTegra None RNAshell RNAstable
   Analyte Extraction and Purification	Nucleic acid digestion	RNAse added None
   Analyte Extraction and Purification	Analyte purification	No additional purification PicoPure purification
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH Beta-actin HMBS PLAUR IL1B ORM1

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