scholarly journals Effects of processing, storage time, and tissue volume in the retrieval and quality of RNA and the expression level of RNU6

2021 ◽  
Author(s):  
Ines Benedetti ◽  
Laura De León ◽  
Niradiz Reyes
Keyword(s):  
Storage Time ◽  
Inverse Correlation ◽  
Ffpe Tissue ◽  
Expression Level ◽  
Clinical Samples ◽  
Tissue Storage ◽  
Tissue Samples ◽  
Rna Quality ◽  
Ffpe Tissues

Background: Molecular analyses of tumor RNA expression have become widely used both for research and clinical purposes. Tumoral tissue preservation is a critical step to ensure accuracy of molecular-based diagnostics, for which, formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source of clinical samples. MicroRNAs are ideal biomarkers in FFPE-tissues, in whose expression evaluation RNU6 is one of the genes used as a normalizer. Our aim was to determine, in FFPE tissue samples, the effects of length of storage and corresponding volume of each studied sample, on the RNA retrieval, quality and concentration, as well as their correlation to the expression level of RNU6. Methods: Fifty tissue blocks with a mean length of tissue storage of 30 months (SD=±12.07, 95% CI= 27.4-34.3). were included. Total RNA was isolated, absorbance and concentrations were determined and correlated with length of storage and volume of tissue. RT-qPCR for RNU6 was performed and their Ct results were correlated to the same parameters. Results: There was a direct correlation between the concentration and quality of the obtained RNA, and an inverse correlation between the tissue storage time and the RNA quality. The volume of tissue studied was not correlated with the RNA quality or concentration. The RNA quality and the length of tissue storage directly correlated to the RNU6 expression level, while RNA concentration and the volume of tissue studied did not affect it. Conclusions: There is an association between longer FFPE tissue storage time with lower RNA quality and lower RNU6 expression level.

scholarly journals Hubungan Lama Penyimpanan Sampel Arsip Jaringan Dalam Blok Parafin Terfiksasi Formalindengan Kualitas Hasil Ekstraksidna Mitokondria Jaringan

2018 ◽  
Vol 1 (3) ◽  
pp. 157-162
Author(s):  
Zen Hafy ◽  
Veny Larasati ◽  
Riana Sari Puspita ◽  
Novizar S ◽  
Haekal M ◽  
...  
Keyword(s):  
Storage Time ◽  
Molecular Study ◽  
Ffpe Tissue ◽  
Genetic Studies ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Pcr Products ◽  
Ffpe Tissues

Formalin-fixed paraffin-embedded (FFPE) archival tissue presents a readily available resource in molecular study nowadays. The quality of nucleid acid, especially mitoconhdrial DNA (mtDNA) extracted from FFPE tissue could be affected by the storage time. Thus, this study investigated if the FFPE tissue’s storage time had an effect on the quality of the extracted mtDNA at Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. DNA was extracted from 16 randomly selected archival FFPE tissues in Laboratory of Pathology Anatomy, RSUP dr. Mohammad Hoesin Palembang. The samples were grouped based on their storage time (less than 1 year and 1 to 5 yrs.’ old). The isolated DNA from each group was amplified using PCR with two primer pairs specifically designto amplify mtDNA of 320 bp and 142bp length, respectively.  The PCR products were visualized by electrophoresismethod. None of the samples from both groups could be amplified witht the 320bp primers. However, the PCR result of the 149 bp primers showed positive for all of the samples from each study group. The study indicated that the storage time does not affect the quality of mtDNA isolated from the FFPE samples archived in Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. Furthermore, the study showed that the mtDNA extracted from FFPE tissue has been degraded, therefore, the samples are not suitable for genetic studies require a long mtDNA amplicon.

scholarly journals Diagnosis of blackleg from cattle tissue impregnated in common filter paper

2021 ◽  
Vol 51 (8) ◽  
Author(s):  
Julia Pires Espíndola ◽  
Luana D’Avila Farias ◽  
Cláudia Balzan ◽  
Valessa Lunkes Ely ◽  
Agueda Palmira Castagna de Vargas
Keyword(s):  
Filter Paper ◽  
Tissue Type ◽  
Limiting Factor ◽  
Direct Pcr ◽  
Tissue Storage ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Clostridium Chauvoei ◽  
Economical Alternative

ABSTRACT: Blackleg, an acute myonecrosis caused by Clostridium chauvoei, is usually underdiagnosed since the rapid transport of adequate samples for laboratory testing is difficult. This study tested a direct polymerase chain reaction (PCR) technique using common filter paper impregnated with cattle tissue samples obtained from animals suspected with blackleg. Twenty-five samples, belonging to eleven animals from Rio Grande do Sul State, Brazil, were analyzed. The direct PCR technique identified eight positive animals corroborating with results from microbiological culture. Skeletal muscle was the most common tissue type used in this study and when the animal was positive the pathogen was always detected in this tissue. Storage time of the impregnated filter paper at room temperature did not prove to be a limiting factor for the quality of the results indicating that this procedure can be carried out in the field and samples be sent in regular mail. Our results suggested that direct PCR of common filter paper impregnated with cattle tissue is a practical and economical alternative for the diagnosis of blackleg.

scholarly journals Determining the utility of veterinary tissue archives for retrospective DNA analysis

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1996 ◽  
Author(s):  
Firas M. Abed ◽  
Michael J. Dark
Keyword(s):  
Dna Analysis ◽  
Inverse Correlation ◽  
Storage Conditions ◽  
Diagnostic Laboratory ◽  
Pilot Studies ◽  
Tissue Samples ◽  
Future Studies ◽  
Significant Difference ◽  
Ffpe Tissues ◽  
Processing And Storage

Histopathology tissue archives can be an important source of specimens for retrospective studies, as these include samples covering a large number of diseases. In veterinary medicine, archives also contain samples from a large variety of species and may represent naturally-occurring models of human disease. The formalin-fixed, paraffin-embedded (FFPE) tissues comprising these archives are rich resources for retrospective molecular biology studies and pilot studies for biomarkers, as evidenced by a number of recent publications highlighting FFPE tissues as a resource for analysis of specific diseases. However, DNA extracted from FFPE specimens are modified and fragmented, making utilization challenging. The current study examines the utility of FFPE tissue samples from a veterinary diagnostic laboratory archive in five year intervals from 1977 to 2013, with 2015 as a control year, to determine how standard processing and storage conditions has affected their utility for future studies. There was a significant difference in our ability to obtain large amplicons from samples from 2015 than from the remaining years, as well as an inverse correlation between the age of the samples and product size obtainable. However, usable DNA samples were obtained in at least some of the samples from all years tested, despite variable storage, fixation, and processing conditions. This study will help make veterinary diagnostic laboratory archives more useful in future studies of human and veterinary disease.

scholarly journals Multiplex Immunoassays of Peptide Hormones Extracted from Formalin-Fixed, Paraffin-Embedded Tissue Accurately Subclassify Pituitary Adenomas

2012 ◽  
Vol 58 (2) ◽  
pp. 366-374 ◽  
Author(s):  
Frederick G Strathmann ◽  
Grace Borlee ◽  
Donald E Born ◽  
Luis F Gonzalez-Cuyar ◽  
Bertrand R Huber ◽  
...  
Keyword(s):  
Protein Expression ◽  
Pituitary Adenomas ◽  
Ffpe Tissue ◽  
Clinical Samples ◽  
Pituitary Neoplasms ◽  
Tissue Samples ◽  
Quantitative Classification ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract BACKGROUND The current gold standard for diagnostic classification of many solid-tissue neoplasms is immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded (FFPE) tissue. Although IHC is commonly used, there remain important issues related to preanalytic variability, nonstandard methods, and operator bias that may contribute to clinically significant error. To increase the quantitative accuracy and reliability of FFPE tissue–based diagnosis, we sought to develop a clinical proteomic method to characterize protein expression in pathologic tissue samples rapidly and quantitatively. METHODS We subclassified FFPE tissue from 136 clinical pituitary adenoma samples according to hormone translation with IHC and then extracted tissue proteins and quantified pituitary hormones with multiplex bead-based immunoassays. Hormone concentrations were normalized and compared across diagnostic groups. We developed a quantitative classification scheme for pituitary adenomas on archived samples and validated it on prospectively collected clinical samples. RESULTS The most abundant relative hormone concentrations differentiated sensitively and specifically between IHC-classified hormone-expressing adenoma types, correctly predicting IHC-positive diagnoses in 85% of cases overall, with discrepancies found only in cases of clinically nonfunctioning adenomas. Several adenomas with clinically relevant hormone-expressing phenotypes were identified with this assay yet called “null” by IHC, suggesting that multiplex immunoassays may be more sensitive than IHC for detecting clinically meaningful protein expression. CONCLUSIONS Multiplex immunoassays performed on FFPE tissue extracts can provide diagnostically relevant information and may exceed the performance of IHC in classifying some pituitary neoplasms. This technique is simple, largely amenable to automation, and likely applicable to other diagnostic problems in molecular pathology.

scholarly journals High-Throughput Simultaneous Analysis of RNA, Protein, and Lipid Biomarkers in Heterogeneous Tissue Samples

2011 ◽  
Vol 57 (11) ◽  
pp. 1545-1555 ◽  
Author(s):  
Vladimír Reiser ◽  
Ryan C Smith ◽  
Jiyan Xue ◽  
Marc M Kurtz ◽  
Rong Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Tissue Sample ◽  
Protein Composition ◽  
Analytical Techniques ◽  
Clinical Samples ◽  
Tissue Samples ◽  
Heterogeneous Tissue

BACKGROUND With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD). METHODS We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured. RESULTS The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting. CONCLUSIONS The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

scholarly journals Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue

2008 ◽  
Vol 56 (11) ◽  
pp. 1033-1042 ◽  
Author(s):  
Joon-Yong Chung ◽  
Till Braunschweig ◽  
Reginald Williams ◽  
Natalie Guerrero ◽  
Karl M. Hoffmann ◽  
...  
Keyword(s):  
Histopathological Examination ◽  
Ffpe Tissue ◽  
Quality Factors ◽  
Rna Quality ◽  
Tissue Processing ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
The Impact ◽  
Formalin Fixed

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphate-buffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at www.jhc.org . Please visit this article online to view these materials.

scholarly journals Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

2012 ◽  
Vol 32 (8) ◽  
pp. 715-720 ◽  
Author(s):  
Paula R. Almeida ◽  
Caroline P. Andrade ◽  
Laura L. Almeida ◽  
Luiz G.S. Oliveira ◽  
Luiza A. Castro ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Mycoplasma Hyopneumoniae ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Histological Features ◽  
Frozen Tissue ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Targeted and nontargeted metabolomics analysis for determining the effect of storage time on the metabolites and taste quality of keemun black tea

2021 ◽  
Vol 359 ◽  
pp. 129950
Author(s):  
Ai Huang ◽  
Zongde Jiang ◽  
Meng Tao ◽  
Mingchun Wen ◽  
Zhipeng Xiao ◽  
...  
Keyword(s):  
Storage Time ◽  
Black Tea ◽  
Taste Quality ◽  
Metabolomics Analysis

scholarly journals Physical Characteristics and Microbial Quality of Ostrich Meat in Relation to the Type of Packaging and Refrigerator Storage Time

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3445
Author(s):  
Olaf K. Horbańczuk ◽  
Artur Jóźwik ◽  
Jarosław Wyrwisz ◽  
Joanna Marchewka ◽  
Agnieszka Wierzbicka
Keyword(s):  
Storage Time ◽  
Modified Atmosphere Packaging ◽  
Physical Characteristics ◽  
Modified Atmosphere ◽  
Packaging System ◽  
Initial Ph ◽  
Viable Bacteria ◽  
L Value ◽  
Meat Samples

The aim of this study was to evaluate the effect of the packaging system type on the physical characteristics and microbial changes in ostrich meat during refrigerated storage. The applied packaging systems were vacuum packaging (VP) and modified atmosphere packaging (MAP) using two combinations of gases: MAP1 (40% O2/40% CO2/20% N2) and MAP2 (60% O2/30% CO2/10% N2). Eight meat samples were obtained in three replicates for all parameters, except for pH, for which six replicates were obtained from the M. ilifibularis (IF) muscle, and were stored in a refrigerator at 2 °C and analyzed at 0, 4, 8, 12 and 16 days for the effect of packaging methods on physical meat quality. The initial pH (5.99) decreased at the end of the storage time for MAP1 to 5.81, whereas VP was stable from day 0 to 12 and increased up to 6.08 on day 16. Regarding meat color, the L* value increased during storage for MAP1 and MAP2 from 36.99 to 40.75 and 41.60, respectively, whereas it declined for VP to 34.22. The same tendencies were reported for redness (a*) and yellowness (b*). Drip loss was the lowest in MAP1 and highest in VP. The lowest total viable bacteria counts were identified in VP, as compared to MAP1 and MAP2.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

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