Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples

Wimmer, Isabella; Tröscher, Anna R.; Brunner, Florian; Rubino, Stephen; Bien, Christian G.; Weiner, Howard L.; Lassmann, Hans; Bauer, Jan

doi:10.1038/s41598-018-24781-6

Cited by 80 publications

(72 citation statements)

References 37 publications

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“…All three genes showed the same expression pattern, being FFPE the samples with significant higher Cq values followed by SF, RNL and SF-OCT, respectively. These results are in agreement with those presented by Wimmer et al 19 , who also observed a clear separation between SF and FFPE samples, both in different Bioanalyzer profiles and the higher Cq values observed with GAPDH, G6PD and SDHA reference genes. Therefore, we present reproducible results even though we tested different reference genes.…”

Section: Discussionsupporting

confidence: 93%

“…It is known that RNA derived from FFPE samples has undergone cross-links, chemical modifications and degradation over time due to the fixation method 17 . These molecular changes support that better quality is usually determined in RNA derived from frozen in comparison with FFPE samples 18,19 although in some cases RNA derived from FFPE was reported to be suitable for gene expression experiments 20 . Today, there are alternative choices for long-term storage of human tissue samples, including RNA preservation solutions.…”

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confidence: 70%

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Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Esteva-Socias

Gómez-Romano

Carrillo-Ávila

et al. 2020

Sci Rep

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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.diluted cDNA and 0,6 µl of PCR-grade water. All PCRs were performed in triplicate from one sample taken from each preservation condition in all 20 patients included. PCR reactions were carried out in 96 well plates and using the CFX96 Real-Time PCR Detection System (Bio-Rad) following the steps: 95 °C for 2 minutes, 40 cycles of amplification (denaturation at 95 °C for 5 seconds, annealing for 10 seconds, and extension at 72 °C for 20 seconds) and final melt curve analysis from 65 °C to 95 °C to exclude unspecific PCR products.The reproducibility and dynamic ranges for qPCR assays were determined by constructing a standard curve, beginning with 100 ng of RNA template as initial concentration and then serial diluted 1/5 until five linear concentration points. For Cq values acquisition, RFU threshold value was adjusted to 50 RFUs for each run.Statistics. All data generated from integrity and functional studies of RNA samples were analysed using multiple comparison tests: ANOVA test with Bonferroni correction to detect significant differences between procedures with at least a signification of p value <0.01. The spearman r index between RNA integ...

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Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 70%

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Esteva-Socias

Gómez-Romano

Carrillo-Ávila

et al. 2020

Sci Rep

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“…The DV200, a quality parameter for RNA derived from FFPE [54], was calculated using the 2100 Expert software (Supplementary Table 1) (version B.02.08.SI648 (SR2)).…”

Section: Methodsmentioning

confidence: 99%

Microglial nodules provide the environment for pathogenic T cells in human encephalitis

et al. 2019

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Microglia nodule formation is a common feature in inflammatory brain diseases mediated by T lymphocytes such as viral and paraneoplastic encephalitis, multiple sclerosis, and Rasmussen encephalitis (RE). However, its role has not been fully understood yet. We hypothesized that, in RE, microglial nodules provide an environment for the initiation of the later dominating T-cell cytotoxicity. In RE stage 0, small primary microglia nodules could be identified in the absence of T cells. These primary nodules showed inflammasome activation and endosomal Toll-like receptor upregulation. In stage 1, T cells migrate into the parenchyma and intermingle with microglial cells, thereby forming secondary nodules in which neurons are destroyed. Whole-genome transcriptome analysis at this point showed upregulation of several inflammatory pathways including interferon signaling and major histocompatibility complex-I signaling. Inflammatory profiles, like the ones observed in RE, could be induced upon TLR3 stimulation in neonatal microglial cell cultures. Taken together, our results point towards activation of endosomal TLRs, resulting in increased interferon signaling, inflammasome activation, and chemokine upregulation as early steps in RE pathogenesis. This activity sets the scene for subsequent infiltration of T cells and destruction of neurons. Similar to RE, this microglial microenvironment might be a crucial step in other T-cell-mediated inflammatory brain diseases.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-01958-5) contains supplementary material, which is available to authorized users.

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“…Cardiac SOD activity was measured colormetrically at 420 nm by the method that is based on the inhibition of pyrogallol autoxidation by SOD, and the results were expressed as U/g tissue [31]. [33]. The SYBR green data were analyzed with a relative quantification to GAPDH as a reference gene.…”

Section: Evaluation Of Cardiac Oxidative and Nitrosative Stressmentioning

confidence: 99%

Paeonol Attenuates Methotrexate-Induced Cardiac Toxicity in Rats by Inhibiting Oxidative Stress and Suppressing TLR4-Induced NF-κB Inflammatory Pathway

Al‐Taher

Morsy

Rifaai

et al. 2020

Mediators of Inflammation

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Methotrexate (MTX) is a commonly used chemotherapeutic agent. Oxidative stress and inflammation have been proved in the development of MTX toxicity. Paeonol is a natural phenolic compound with various pharmacological activities including antioxidant and anti-inflammatory properties. The aim of the present study was to evaluate the protective effect of paeonol against MTX-induced cardiac toxicity in rats and to evaluate the various mechanisms that underlie this effect. Paeonol (100 mg/kg) was administered orally for 10 days. MTX cardiac toxicity was induced at the end of the fifth day of the experiment, with or without paeonol pretreatment. MTX-induced cardiac damage is evidenced by a distortion in the normal cardiac histological structure, with significant oxidative and nitrosative stress shown as a significant increase in NADPH oxidase-2, malondialdehyde, and nitric oxide levels along with a decrease in reduced glutathione concentration and superoxide dismutase activity compared to the control group. MTX-induced inflammatory effects are evidenced by the increased cardiac toll-like receptor 4 (TLR4) mRNA expression and protein level as well as increased cardiac tumor necrosis factor- (TNF-) α and interleukin- (IL-) 6 levels along with increased nuclear factor- (NF-) κB/p65 immunostaining. MTX increased apoptosis as shown by the upregulation of cardiac caspase 3 immunostaining. Paeonol was able to correct the oxidative and nitrosative stress as well as the inflammatory and apoptotic parameters and restore the normal histological structure compared to MTX alone. In conclusion, paeonol has a protective effect against MTX-induced cardiac toxicity through inhibiting oxidative and nitrosative stress and suppressing the TLR4/NF-κB/TNF-α/IL-6 inflammatory pathway, as well as causing an associated reduction in the proapoptotic marker, caspase 3.

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Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples

Cited by 80 publications

References 37 publications

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples

Microglial nodules provide the environment for pathogenic T cells in human encephalitis

Paeonol Attenuates Methotrexate-Induced Cardiac Toxicity in Rats by Inhibiting Oxidative Stress and Suppressing TLR4-Induced NF-κB Inflammatory Pathway

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