Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues

Lv, Yubao; Li, Shiying; Li, Zhihong; Tao, Ruiyang; Shao, Yu; Chen, Yijiu

doi:10.1007/s00414-019-02210-1

Cited by 9 publications

(8 citation statements)

References 70 publications

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“…Secondly, our study design did not include a comparison between fixed and non-fixed tissue sample to test the limitations of the FFPE extraction step. However, the available literature reports comparing paired fresh and FFPE tissues show that even though RNA integrity can decrease during FFPE extraction, reproducible and highly correlated gene expression data can be obtained from FFPE samples of various tissue types, including breast cancer subtypes [32][33][34][35]. In addition, the use of more than one representative cell line for each breast cancer subtype would have been ideal.…”

Section: Discussionmentioning

confidence: 99%

Long Non-Coding RNA Expression in Laser Micro-Dissected Luminal A and Triple Negative Breast Cancer Tissue Samples—A Pilot Study

et al. 2021

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Background and Objectives: Breast cancer (BC) remains one of the major causes of cancer death in women worldwide. The difficulties in assessing the deep molecular mechanisms involved in this pathology arise from its high complexity and diverse tissue subtypes. Long non-coding RNAs (lncRNAs) were shown to have great tissue specificity, being differentially expressed within the BC tissue subtypes. Materials and Methods: Herein, we performed lncRNA profiling by PCR array in triple negative breast cancer (TNBC) and luminal A tissue samples from 18 BC patients (nine TNBC and nine luminal A), followed by individual validation in BC tissue and cell lines. Tissue samples were previously archived in formalin-fixed paraffin-embedded (FFPE) samples, and the areas of interest were dissected using laser capture microdissection (LCM) technology. Results: Two lncRNAs (OTX2-AS1 and SOX2OT) were differentially expressed in the profiling analysis (fold change of 205.22 and 0.02, respectively, p < 0.05 in both cases); however, they did not reach statistical significance in the individual validation measurement (p > 0.05) when analyzed with specific individual assays. In addition, GAS5 and NEAT1 lncRNAs were individually assessed as they were previously described in the literature as being associated with BC. GAS5 was significantly downregulated in both TNBC tissues and cell lines compared to luminal A samples, while NEAT1 was significantly downregulated only in TNBC cells vs. luminal A. Conclusions: Therefore, we identified GAS5 lncRNA as having a differential expression in TNBC tissues and cells compared to luminal A, with possible implications in the molecular mechanisms of the TNBC subtype. This proof of principle study also suggests that LCM could be a useful technique for limiting the sample heterogeneity for lncRNA gene expression analysis in BC FFPE tissues. Future studies of larger cohort sizes are needed in order to assess the biomarker potential of lncRNA GAS5 in BC.

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Section: Discussionmentioning

confidence: 99%

Long Non-Coding RNA Expression in Laser Micro-Dissected Luminal A and Triple Negative Breast Cancer Tissue Samples—A Pilot Study

et al. 2021

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“…Irrespective of the situation with other methods, it is necessary to consider this issue for RT-qPCR measurements of clinical samples. Moreover, the specific problem of FFPE material needs clarification 31 , 62 - 64 . New RNA quality metrics, which are more sensitive than the RIN values generally used up to now, are recommended to define the preanalytical RNA conditions for reliable expression analyses in future studies 63 , 64 .…”

Section: Discussionmentioning

confidence: 99%

“…This might also change the potential degrading activity of endonucleases on total RNA under these conditions. Some studies have noted potential difficulties in quantifying circRNAs in degraded total RNA samples 7 , 13 , 30 , 31 . Therefore, it is surprising that the majority of recent circRNA expression-related studies in cancer tissues have ignored the possible influence of RIN on the measured values ( Table S1 ).…”

Section: Introductionmentioning

confidence: 99%

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality

et al. 2020

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Background: Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions. Methods: Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs ( circEGLN3, circRHOBTB3, circCSNK1G3, circRNA4 , and circRNA9 ), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6. Results: The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6. Conclusions: Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies.

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“…A previous study evaluated the stability of several circRNAs, including circ-AFF1, in the hearts of mice within 8 days after death, and concluded that, as reference genes, circRNAs were more stable than other kinds of RNAs are in dead bodies [ 107 , 108 ]. Studies also reported that circRNAs presented relatively consistent and stable expression profiles in formalin-fixed paraffin-embedded tissues compared with their corresponding linear transcripts [ 128 ]. However, real-world conditions are much more complicated than laboratory conditions, especially with postmortem cases.…”

Section: Circrnas As Novel Biomarkers In Postmortem Scd Diagnosismentioning

confidence: 99%

“…However, real-world conditions are much more complicated than laboratory conditions, especially with postmortem cases. Using short amplicons and standardized RT-qPCR assays in autopsy cases might improve the possibility of performing accurate quantitative analysis of circRNAs [ 128 ]. On the plus side, forensic scientists can obtain sufficient tissue samples through autopsies, even including some unconventional samples such as pericardial fluid, which also expands the conditions for the selection of appropriate biomarkers.…”

Section: Circrnas As Novel Biomarkers In Postmortem Scd Diagnosismentioning

confidence: 99%

Circular RNAs in Sudden Cardiac Death Related Diseases: Novel Biomarker for Clinical and Forensic Diagnosis

2021

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The prevention and diagnosis of sudden cardiac death (SCD) are among the most important keystones and challenges in clinical and forensic practice. However, the diagnostic value of the current biomarkers remains unresolved issues. Therefore, novel diagnostic biomarkers are urgently required to identify patients with early-stage cardiovascular diseases (CVD), and to assist in the postmortem diagnosis of SCD cases without typical cardiac damage. An increasing number of studies show that circular RNAs (circRNAs) have stable expressions in myocardial tissue, and their time- and tissue-specific expression levels might reflect the pathophysiological status of the heart, which makes them potential CVD biomarkers. In this article, we briefly introduced the biogenesis and functional characteristics of circRNAs. Moreover, we described the roles of circRNAs in multiple SCD-related diseases, including coronary artery disease (CAD), myocardial ischemia or infarction, arrhythmia, cardiomyopathy, and myocarditis, and discussed the application prospects and challenges of circRNAs as a novel biomarker in the clinical and forensic diagnosis of SCD.

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Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues

Cited by 9 publications

References 70 publications

Long Non-Coding RNA Expression in Laser Micro-Dissected Luminal A and Triple Negative Breast Cancer Tissue Samples—A Pilot Study

Long Non-Coding RNA Expression in Laser Micro-Dissected Luminal A and Triple Negative Breast Cancer Tissue Samples—A Pilot Study

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality

Circular RNAs in Sudden Cardiac Death Related Diseases: Novel Biomarker for Clinical and Forensic Diagnosis

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