Abstract
Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.
Highlights
Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers
Results obtained from the room temperature (RT)-quantitative Polymerase Chain Reaction (qPCR) and Bioanalyzer 2100 were plotted on three independent graphs and nonparametric Spearman correlation was performed to better elucidate the probable relationship between the two metrics (Fig. 5)
The correlation measured by R Spearman index between Cq mean and RNA Integrity Number (RIN) values was of −0.7379 for HPRT1gene, −0.8011 for SNRPD3 gene and −0.7252 for Jun gene (p < 0.001)
Summary
Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. The analysis and validation of biomarkers in large and homogeneous representative cohorts of biological samples with associated data is required, often only possible thanks to collaborative actions between institutions[1,2] In this scenario, biobanks play a central role in current research, making human biological samples available to researchers, guaranteeing their best quality and associating relevant information to them, integrating clinical, pathological and molecular information and, being a commitment of ethical and legal requirements[3,4]. Despite having identified some of the main pre-analytical contributors to the loss and quality assurance of human biospecimens, there is still not enough clear information available to know to what extent each of these factors contributes Aiming to clarify this gap in knowledge, Stabilization method RNAlater Snap Frozen Snap Frozen-OCT FFPE. This fact is due to the interaction of other pre-analytical factors such as preservation type[12], preservation method[13], long term storage[14,15], RNA extraction methodology[16], among others
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