Assessing the quality of RNA isolated from human breast tissue after ambient room temperature exposure.

High quality human tissue is essential for molecular research, but pre-analytical conditions encountered during tissue collection could degrade tissue RNA. We evaluated how prolonged exposure of non-diseased breast tissue to ambient room temperature (22±1°C) impacted RNA quality. Breast tissue received between 70 to 190 minutes after excision was immediately flash frozen (FF) or embedded in Optimal Cutting Temperature (OCT) compound upon receipt (T0). Additional breast tissue pieces were further exposed to increments of 60 (T1 = T0+60 mins), 120 (T2 = T0+120 mins) and 180 (T3 = T0+180 mins) minutes of ambient room temperature before processing into FF and OCT. Total exposure, T3 (T0+180 mins) ranged from 250 minutes to 370 minutes. All samples (FF and OCT) were stored at -80°C before RNA isolation. The RNA quality assessment based on RNA Integrity Number (RIN) showed RINs for both FF and OCT samples were within the generally acceptable range (mean 7.88±0.90 to 8.52±0.66). No significant difference was observed when RIN at T0 was compared to RIN at T1, T2 and T3 (FF samples, p = 0.43, 0.56, 0.44; OCT samples, p = 0.25, 0.82, 1.0), or when RIN was compared between T1, T2 and T3. RNA quality assessed by quantitative real-time PCR (qRT-PCR) analysis of beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A (CYPA), and porphobilinogen deaminase (PBGD) transcripts showed threshold values (Ct) that indicate abundant and intact target nucleic acid in all samples (mean ranging from 14.1 to 25.3). The study shows that higher RIN values were obtained for non-diseased breast tissue up to 190 minutes after resection and prior to stabilization. Further experimental exposure up to 180 minutes had no significant effect on RIN values. This study strengthens the rationale for assessing RIN and specific gene transcript levels as an objective method for determining how suitable RNA will be for a specific research purpose ("fit-for purpose").

Tissue imprints: assessing their potential for routine biobanking collection.

Biomedical research depends on the availability of good quality biospecimens. Unfortunately, certain specimens are scarce due to disease rarity or size restrictions of surgical materials. To increase access to limited surgical specimens, Biobanks need to reassess and adjust their collection programs. We evaluated the feasibility of adapting "touch imprints" to gain access to limited surgical specimens as well as to maximize the use of "precious" specimens. We utilized 12 kidney samples for touch imprints on defined areas of microscope glass slides and FTA paper. DNA was isolated from glass slides on the day of preparation, Day 0, and from glass slide and FTA paper preparations after two weeks of storage at room temperature and -80°C. Yield and purity of DNA from reference kidney samples were compared to DNA from the touch imprints and the quality determined by real-time PCR using the amplification of Cyclophilin A (Cyc A) as an index. DNA quality for glass slides at Day 0 was not significantly different from DNA after two weeks at room temperature (glass at room temperature; p=0.111 and 0.097, yield and purity, respectively) and after two weeks at -80°C (glass -80°C; p=0.358 and 0.281, yield and purity, respectively). Glass slide DNA at room temperature and -80°C were not significantly different (p=0.795 and 0.146 for yield and purity, respectively). DNA from FTA paper at room temperature and from FTA paper at -80°C were significantly different from glass at room temperature and glass at -80°C (p=0.002, respectively). Threshold values for Cyc A were ≤28 for the reference DNA and ≤32 for DNA from glass and FTA paper. This study demonstrates that touch preparations on microscope glass slides and FTA paper can provide sufficient and good quality DNA suitable for PCR. Touch imprints could therefore be adopted by biobanks to collect and bank biological materials from limited surgical specimens.