TY - JOUR
T1 - Assessing the quality of RNA isolated from human breast tissue after ambient room temperature exposure
AU - Somiari, Stella B.
AU - Shuss, Susan
AU - Liu, Jianfang
AU - Mamula, Kimberly
AU - O’Donnell, Amy
AU - Deyarmin, Brenda
AU - Kane, Jennifer
AU - Greenawalt, Amber
AU - Larson, Caroline
AU - Rigby, Sean
AU - Hu, Hai
AU - Shriver, Craig D.
N1 - Publisher Copyright:
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PY - 2022/1
Y1 - 2022/1
N2 - 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±1C) 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 -80C 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”).
AB - 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±1C) 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 -80C 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”).
UR - http://www.scopus.com/inward/record.url?scp=85123180376&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0262654
DO - 10.1371/journal.pone.0262654
M3 - Article
C2 - 35041696
AN - SCOPUS:85123180376
SN - 1932-6203
VL - 17
JO - PLoS ONE
JF - PLoS ONE
IS - 1 January
M1 - e0262654
ER -