Biobanking Practice: RNA Storage at Low Concentration Affects Integrity

Abstract

A critical issue in defining protocols for biobanking practices is the preservation of total RNA for assessing the whole transcriptome and ensuring that it can be utilized in clinically oriented studies. Storage conditions, such as temperature and the length of time that tissues and purified RNA stay frozen, may directly impact RNA preservation. In this study, we evaluated a) the quality of RNA (as measured by RNA Integrity Number) purified from head and neck tumor tissues stored at -140°C for distinct time intervals of up to 7 years, and b) the quality of their respective RNAs stored for 4 years at -80°C when diluted at either 250 ng/μL or 25 ng/μL, with repeated freezing and thawing. Additionally, we generated a profile of the RNA collection of human tumors from different body sites stored at the AC Camargo Biobank. Our results showed no significant change in RIN values according to length of storage at -140°C. With respect to RNA aliquots stored at -80°C, RNA integrity at 250 ng/μL was preserved, while statistically significant degradation was observed at 25 ng/μL after only 8 months of storage. The RNA collection from most of the human tumors stored at the AC Camargo Biobank exhibited high quality, with average RIN around seven. However, ovary and stomach samples had the greatest RNA degradation. Taken together, the results show that both the temperature of preservation and the concentration of RNA should be strictly controlled by the biobank staff involved in macromolecule purification. Moreover, the RNAs from our biobank can be useful for the most demanding methods of gene expression analysis by virtue of adherence to optimal standard operating procedures for both tissue and macromolecule laboratories.

Preanalytical Variables Affecting the Integrity of Human Biospecimens in Biobanking

“…Preanalytical storage factors affecting analyte quantity, quality, or gene expression are temperature, storage time, concentration of RNA, and repeated thaws (53,119,120 ). As for RNA, new technologies for dry storage at room temperature have been developed (121)(122)(123).…”

Impact of in Vivo Ischemic Time on RNA Quality—Experiences from a Colon Cancer Biobank

“…16,17 Whether tissue should be stored at -80°C or -140°C remains unclear. 13,23 We observed no adverse effects on RNA quality in fresh-frozen tissue after storage at -80°C for up to 6 years.…”

Tumor banking for health research in Brazil and Latin America: time to leave the cradle

“…In theory, tissues that are quickly frozen and kept below this temperature are free of any degradation of macromolecules (e.g., hydrolysis or enzymatic degradation) [9][10][11]. In a recent study, our group showed no significant change in RIN (RNA Integrity Number) values of cryopreserved tissues stored at −140°C for up to 7 years [12]. On the other hand, researchers at the Indiana University, examining sets of samples that were stored at −80°C observed that all samples with less than 18 months of storage had high-quality RNA (also analyzed by RIN), compared to only 48% of the samples that were stored for longer periods (>8 years) [8].…”

“…In the same study in which our group analyzed the storage conditions of tissues, the long-term preservation of RNA molecules was also addressed. With respect to RNA aliquots (which are stored at −80°C), our results shown that the concentration of stored RNA strongly affects its integrity in long-term storage, indicating that biobanks that intend to distribute not only tissues, but also extracted RNA macromolecules for research must also control this variable, storing RNA in high concentration (>200 ng/μl) [12].…”

“…On the other hand, researchers at the Indiana University, examining sets of samples that were stored at −80°C observed that all samples with less than 18 months of storage had high-quality RNA (also analyzed by RIN), compared to only 48% of the samples that were stored for longer periods (>8 years) [8]. Currently, there is no published study that has compared the quality of macromolecules extracted from tissues that were submitted to the same collection and cryopreservation protocols and stored at −140 and −80°C, so biobanks that intend to store tissues for long periods of time should consider adopt temperatures below −130°C (either using cryogenic freezers or liquid nitrogen containers) [11,12].…”