RT Journal Article
T1 Impact of different stabilization methods on RT-qPCR results using human lung tissue samples.
A1 Esteva-Socias, Margalida
A1 Gomez-Romano, Fernando
A1 Carrillo-Avila, Jose Antonio
A1 Sanchez-Navarro, Alicia Loreto
A1 Villena, Cristina
K1 Reverse transcription polymerase chain reaction
K1 PCR-based techniques
K1 Biomarkers
K1 Eukaryote
K1 Gene expression analysis
AB 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.
PB Nature Publishing Group
YR 2020
FD 2020-01-20
LK http://hdl.handle.net/10668/15177
UL http://hdl.handle.net/10668/15177
LA en
NO Esteva-Socias M, Gómez-Romano F, Carrillo-Ávila JA, Sánchez-Navarro AL, Villena C. Impact of different stabilization methods on RT-qPCR results using human lung tissue samples. Sci Rep. 2020 Feb 27;10(1):3579.
NO We thank all volunteers of Pulmonary Biobank Consortium for the participation and sample donation. We also thank Institut d'Investigació Sanitària de les Illes Balears (IdISBa) for publication funding (Liberi 2020 Program). This work was funded by the Ministerio de Ciencia, Innovación y Universidades of Spain and Instituto de Salud Carlos III (PI16/00528) through the Spanish Biobank Network (PT17/0015/0001) and granted by Conselleria d’Innovació, Recerca i Turisme del Govern de les Illes Balears (TEC/002/2017).
DS RISalud
RD Apr 17, 2025