[2017] Effect of the Duration of Cold Ischemic Time and Temperature Conditions on Maintenance of RNA Integrity Number (RIN) in Biospecimens

Gabriel Sica, Charles Butler, Guojing Zhang, Shishir Maithel, Michael Rossi, Taofeek Owonikoko. Emory University, Atlanta, GA

Background: The RIN determines the integrity of extracted RNA by analyzing RNA using a combination of micropapillary electrophoresis and fluorescence detection that is analyzed using an established software algorithm. The RIN is a numerical scale ranging from 1(degraded) to 10(intact). A RIN >7 is considered sufficiently intact for most applications and is affected by ischemic time. Ischemic time can be separated into two components, warm ischemic time (surgical incision to specimen removal) and cold ischemic time (resection to tissue stabilization e.g. freezing). Previous reports have indicated conflicting results on the effect of cold ischemic time on RIN and whether temporary storage of specimens on ice prior to freezing results in improved RIN.
Design: Resected human pancreatic tissues were stored at 4ºC prior to tissue stabilization. For the murine xenotransplant studies, athymic nude mice bearing small cell lung carcinoma (SCLC) xenografts were sacrificed by cervical dislocation at day 14 post inoculation when tumor volume averaged 800 mm3. Tumors were collected and dissected into 2 mm pieces and either frozen immediately, incubated at room temperature or placed on ice for the indicated time period prior to freezing. All samples were snap frozen in liquid nitrogen and stored at -80ºC. Total RNA was extracted using miRNeasy kit, concentration was determined using a Nanodrop spectrophotometer and RNA was analyzed using an Agilent 2100 bioanalyzer Nano LapChip.
Results: The RIN for pancreas tissues ranged from 1.7 to 8.1 and did not correlate with the length of time the tissue was stored at 4ºC prior to freezing.

Table 1. Pancreas Tissue
SpecimenTime to StabilizationRIN
145 minutes1.7
22.5 hours3.1
36 hours8.1
412 hours5.6
512 hours2.3


In the harvested xenografted SCLC tissue, RIN ranged from 8.4 to 10 with no differences identified between tissues immediately snap frozen, stored at room temperature or on ice prior to long term freezing.

Table 2. Small Cell Carcinoma Xenotransplant RIN Values When Incubated on Ice or Room Temperature (RT)
 RIN - mouse 1RIN - mouse 2
Time to Stabilization (Minutes)IceRTIceRT
010 10 
1510109.89.6
3010109.39.6
609.99.79.88.4



Conclusions: Cold ischemic time up to 60 minutes at room temperature or on ice did not impact RIN values. Our result suggests that this factor may not be the main preanalytic variable determining the RIN of fresh frozen tissues. Previous recommendations for stabilization of pancreas tissues on ice or at 4ºC prior to freezing was partly supported by our results.
Category: Quality Assurance

Tuesday, March 5, 2013 9:30 AM

Poster Session III # 285, Tuesday Morning

 

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