RNA quality in fresh frozen prostate tissue from patients operated with radical prostatectomy

Abstract

Background: High-throughput technologies such as microarray have enhanced the discovery of new biomarkers in prostate cancer. However, the reliability of transcriptome analyses is limited by the RNA quality. Objective: Identification of variables influencing the RNA quality in radical prostatectomy specimens. Material and methods: RNA was extracted using an automatic extraction method for 354 samples from 38 fresh frozen prostate slices, and by manual extraction for 28 samples from 5 slices. RNA quality was measured using the RIN method (RNA Integrity Number). Evaluation of tissue composition was performed by light-microscopy for each sample. Age, total operative time, estimated blood loss, prostate volume, prostate specific antigen (s-PSA) and postoperative Gleason score were registered. The independent variables were correlated to the RIN score in a multiple linear regression model, taking p < 0.05 as the significance limit. Results: The amount of blood loss during prostatectomy and the amount of stroma in the tissue sample both correlated negatively with the RIN score (p = 0.03 and 0.02). Automatically extracted samples which were exposed to heat according to the RNA extraction protocol, had lower mean RNA quality (5.5, 1.46 SD) than manually extracted samples, not exposed to heat (8.7, 0.86 SD), suggesting degradation by temperature sensitive RNases, mainly residing in the stroma. Conclusion: The highest RNA quality isolated by an automatic method from fresh frozen prostate tissue is obtained from patients with low peroperative blood loss and from samples with a low stromal fraction.

Key Words::

• Cryopreservation
• molecular diagnostic techniques
• oligonucleotide array sequence analysis
• ribonucleases
• RNA stability

Acknowledgements

The authors thank Eli Helge and Toril Rolfseng for excellent assistance with the laboratory work, Jørn-Ove Sæternes for the design and development of equipment used to harvest and handle frozen prostate tissue, Marius Dybwad for assistance with cell line culture and Jan Gerhard Mjønes for an essential contribution to the material collection of tissue material.

The study was granted with 60 000 NOK from the Cancer Foundation of St. Olavs Hospital, University Hospital of Trondheim, Norway.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.