Assessing RNA integrity by digital RT-PCR: Influence of extraction, storage, and matrices.

The development of high-throughput sequencing has greatly improved our knowledge of microbial diversity in aquatic environments and its evolution in highly diverse ecosystems. Relevant microbial diversity description based on high-throughput sequencing relies on the good quality of the nucleic acid recovered. Indeed, long genetic fragments are more informative for identifying mutation combinations that characterize variants or species in complex samples. This study describes a new analytical method based on digital Polymerase Chain Reaction (PCR) partitioning technology for assessing the fragmentation of nucleic acid and more specifically viral RNA. This method allows us to overcome limits associated with hydrolysis probe-based assay by focusing on the distance between different amplicons, and not, as usual, on the size of amplicons. RNA integrity can thus be determined as a new fragmentation index, the so-called Fragment size 50. The application of this method has provided information on issues that are inherent in environmental analyses, such as the storage impact of raw samples or extracted RNA, extraction methods, and the nature of the sample on the integrity of viral RNA. Finally, the estimation of fragment size by digital PCR (dPCR) showed a very strong similarity with the fragment size sequenced using Oxford Nanopore Technology. In addition to enabling objective improvements in analytical methods, this approach could become a systematic quality control prior to any long-read sequencing, avoiding insufficiently productive sequencing runs or biases in the representativeness of sequenced fragments.

Screening of NIAS World Rice Core Collection for Seeds with Long Longevity as Useful Potential Breeding Materials Focusing on the Stability of Embryonic RNAs.

Seed longevity is a crucial trait for the seed industry and genetic resource preservation. To develop excellent cultivars with extended seed lifespans, it is important to understand the mechanism of keeping seed germinability long term and to find useful genetic resources as prospective breeding materials. This study was conducted to identify the best cultivars with a high and stable seed longevity trait in the germplasm of rice (Oryza sativa L.) and to analyze the correlation between seed longevity and embryonic RNA integrity. Seeds from 69 cultivars of the world rice core collection selected by the NIAS in Japan were harvested in different years and subjected to long-term storage or controlled deterioration treatment (CDT). The long-term storage (4 °C, RH under 35%, 10 years) was performed on seeds harvested in 2010 and 2013. The seeds harvested in 2016 and 2019 were used for CDT (36 °C, RH of 80%, 40 days). Seed longevity and embryonic RNA integrity were estimated by a decrease in the germination percentage and RNA integrity number (RIN) after long-term storage or CDT. The RIN value was obtained by the electrophoresis of the total RNA extracted from the seed embryos. Seeds of "Vandaran (indica)", "Tupa 729 (japonica)", and "Badari Dhan (indica)" consistently showed higher seed longevity and embryonic RNA integrity both under long-term storage and CDT conditions regardless of the harvest year. A strong correlation (R2 = 0.93) was observed between the germination percentages and RIN values of the seeds after the long-term storage or CDT among nine cultivars selected based on differences in their seed longevity. The study findings revealed the relationship between rice seed longevity and embryo RNA stability and suggested potential breeding materials including both japonica and indica cultivars for improving rice seed longevity.

Effect of Different Staining Methods on Brain Cryosections.

Staining frozen sections is often required to distinguish cell types for spatial transcriptomic studies of the brain. The impact of the staining methods on the RNA integrity of the cells becomes one of the limitations of spatial transcriptome technology with microdissection. However, there is a lack of systematic comparisons of different staining modalities for the pretreatment of frozen sections of brain tissue as well as their effects on transcriptome sequencing results. In this study, four different staining methods were analyzed for their effect on RNA integrity in frozen sections of brain tissue. Subsequently, differences in RNA quality in frozen sections under different staining conditions and their impact on transcriptome sequencing results were assessed by RNA-seq. As one of the most commonly used methods for staining pathological sections, HE staining seriously affects the RNA quality of frozen sections of brain tissue. In contrast, the homemade cresyl violet staining method developed in this study has the advantages of short staining time, low cost, and less RNA degradation. The homemade cresyl violet staining proposed in this study can be applied instead of HE staining as an advance staining step for transcriptome studies in frozen sections of brain tissue. In the future, this staining method may be suitable for wide application in brain-related studies of frozen tissue sections. Moreover, it is expected to become a routine step for staining cells before sampling in brain science.

RNA quality score evaluation: A preliminary study of RNA integrity number (RIN) and RNA integrity and quality number (RNA IQ).

In the past several years, with the in-depth development of RNA-related research, exploring the application of transcriptome and corresponding RNA biomarkers has become one of the research hotspots in the field of forensic science. High-quality RNA is essential for successful downstream workflows, especially in the steps of screening biomarkers by microarray or RNA sequencing (RNA-seq). Thus, accurately evaluating the quality of RNA samples is a critical step in obtaining meaningful expression data. The RNA integrity number (RIN) generated from the Agilent Bioanalyzer system has been widely used for RNA quality control in the past two decades. Recently, Thermo Fisher Scientific launched a ratiometric fluorescence-based method to quickly check whether an RNA sample has degraded, and the results are presented as RNA integrity and quality number (RNA IQ). Both quality score systems determine RNA quality using a numerical system based on a scale of 1-10, with 1 denoting significantly degraded specimens and 10 representing high-quality, intact RNA samples. In this preliminary study, we evaluated the consistency, reproducibility and linearity of two quality scores in RNA quality determination by analyzing heat- and RNase- artificially degraded samples. Meanwhile, the expression levels of three microRNAs (hsa-let-7 g-5p, hsa-miR-93-5p and hsa-miR-191-5p) in intact and severely degraded RNA samples were estimated by TaqMan-qPCR and droplet digital PCR. Overall, both quality scores showed good repeatability and reproducibility in their respective tests. In the samples subjected to thermal degradation, RIN showed a trend corresponding to heating time, while RNA IQ value showed almost no change on the time gradient. However, in RNase A mediated degradation, RNA IQ value observed better linearity. Furthermore, the expression levels of three microRNAs in the severely degraded samples did not show significant changes compared to the intact RNA samples. RNA degradation is a very complex and highly variable process, which is difficult to comprehensively evaluate through any one index and cannot directly compare these two parameters. Nevertheless, combined with previous research results and the expression levels of three microRNAs in this study, analyzing RNA biomarkers with stable regions or small sizes in challenged samples may be a conservative and reliable approach.

An Optimized and Cost-Effective RNA Extraction Method for Secondary Metabolite-Enriched Tissues of Norway Spruce (Picea abies).

Since the development of next-generation sequencing techniques and with the growing interest in transcriptomic studies, there is a demand for high-throughput RNA extraction techniques. General RNA extraction protocols are unreliable when it comes to the quality and quantity of isolated RNA obtained from different tissue types of different plant species. Despite Norway spruce (Picea abies) being one of the most significant and commercially valuable tree species in European forests, only limited genetic research is available. In this study, we developed a cetyltrimethylammonium bromide (CTAB) protocol by modifying the original method. We compared this CTAB protocol with other widely used methods for extracting RNA from different tissues (needle, phloem, and root) of Norway spruce, known for its richness in polyphenols, polysaccharides, and secondary metabolites. The modified CTAB method proves to be superior to the kit-based and TRIzol-based methods for extracting RNA from the metabolite-rich tissues of Norway spruce, resulting in high RNA quality and integrity values (RIN~7-9). The modified CTAB RNA extraction method is rapid, cost-effective, and relatively simple in yielding the desired RNA quality from Norway spruce tissues. It is optimal for RNA sequencing and other downstream molecular applications.

Direct specimen collection during routine operation improves nucleic acid quality in genetic analysis samples for head and neck tumors: A retrospective study.

The nucleic acid integrity of head and neck squamous cell carcinoma (HNSCC) samples is poor, and the material available for genetic analysis is limited. Therefore, to expand the effectiveness of personalized medicine in patients with HNSCC, a new sampling method is needed. In total, 128 samples from 44 patients with HNSCC were studied: 32 genetic analysis samples (GASs) collected as 5 × 5 × 5 mm tissue fragments from resected large tumors and immediately embedded in a small formalin bottle within 10 min (i.e., the ischemic time), 43 primary tumor components (primary), 14 decalcified tumor (DC) samples, 32 metastatic tumors in lymph nodes (LNs), and 7 parakeratinized components (PKCs). The nucleic acid quality in the GAS, primary, DC, LN, and PKC groups was compared and next-generation sequencing (NGS) was performed. DNA integrity number and percentage of RNA fragments with > 200 nucleotides were significantly higher in the GAS group than those in the other groups. RNA integrity number decreased first in LN, followed by GAS, primary, and DC. No significant differences were observed in DIN, RIN and DV200 among the PKC, primary and LN. Following methyl green-pyronin staining, preserved DNA and RNA were not visualized in DC samples. Most NGS metrics did not differ significantly among primary, LN, and PKC samples. In conclusion, GASs should be collected during routine hospital activities. When the volume of viable materials is limited, PKCs should be considered for genetic analysis.

Measuring Up: A Comparison of TapeStation 4200 and Bioanalyzer 2100 as Measurement Tools for RNA Quality in Postmortem Human Brain Samples.

The determination of RNA integrity is a critical quality assessment tool for gene expression studies where the experiment's success is highly dependent on the sample quality. Since its introduction in 1999, the gold standard in the scientific community has been the Agilent 2100 Bioanalyzer's RNA integrity number (RIN), which uses a 1-10 value system, from 1 being the most degraded, to 10 being the most intact. In 2015, Agilent launched 4200 TapeStation's RIN equivalent, and reported a strong correlation of r2 of 0.936 and a median error < ±0.4 RIN units. To evaluate this claim, we compared the Agilent 4200 TapeStation's RIN equivalent (RINe) and DV200 to the Agilent 2100 Bioanalyzer's RIN for 183 parallel RNA samples. In our study, using RNA from a total of 183 human postmortem brain samples, we found that the RIN and RINe values only weakly correlate, with an r2 of 0.393 and an average difference of 3.2 RIN units. DV200 also only weakly correlated with RIN (r2 of 0.182) and RINe (r2 of 0.347). Finally, when applying a cut-off value of 6.5 for both metrics, we found that 95.6% of samples passed with RIN, while only 23.5% passed with RINe. Our results suggest that even though RIN (Bioanalyzer) and RINe (TapeStation) use the same 1-10 value system, they should not be used interchangeably, and cut-off values should be calculated independently.

Swiss Vascular Biobank: Evaluation of Optimal Extraction Method and Admission Solution for Preserving RNA from Human Vascular Tissue.

Proper biobanking is essential for obtaining reliable data, particularly for next-generation sequencing approaches. Diseased vascular tissues, having extended atherosclerotic pathologies, represent a particular challenge due to low RNA quality. In order to address this issue, we isolated RNA from vascular samples collected in our Swiss Vascular Biobank (SVB); these included abdominal aortic aneurysm (AAA), peripheral arterial disease (PAD), healthy aorta (HA), and muscle samples. We used different methods, investigated various admission solutions, determined RNA integrity numbers (RINs), and performed expression analyses of housekeeping genes (ACTB, GAPDH), ribosomal genes (18S, 28S), and long non-coding RNAs (MALAT1, H19). Our results show that RINs from diseased vascular tissue are low (2-4). If the isolation of primary cells is intended, as in our SVB, a cryoprotective solution is a better option for tissue preservation than RNAlater. Because RNA degradation proceeds randomly, controls with similar RINs are recommended. Otherwise, the data might convey differences in RNA degradation rather than the expressions of the corresponding genes. Moreover, since the 18S and 28S genes in the diseased vascular samples were degraded and corresponded with the low RINs, we believe that DV200, which represents the total RNA's disintegration state, is a better decision-making aid in choosing samples for omics analyses.

Optimisation of Sample Preparation from Primary Mouse Tissue to Maintain RNA Integrity for Methods Examining Translational Control.

The protein output of different mRNAs can vary by two orders of magnitude; therefore, it is critical to understand the processes that control gene expression operating at the level of translation. Translatome-wide techniques, such as polysome profiling and ribosome profiling, are key methods for determining the translation rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions, which need to be avoided when maintaining the interaction and position of the ribosome with the mRNA is required. Here, we detail the cell lysis conditions that produce high-quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue.

The Influences of Cryopreservation Methods on RNA, Protein, Microstructure and Cell Viability of Skeletal Muscle Tissue.

Background: Different experiments require different sample storage methods. The commonly used preservation methods in biobank practice cannot fully meet the multifarious requirements of experimental techniques. Programmable controlled slow freezing (PCSF) can maintain the viability of tissue. In this study, we hypothesized that PCSF-preserved samples have potential advantages in matching subsequent experiments compared with existing methods. Methods: We compared the differences on skeletal muscle tissue RNA integrity, protein integrity, microstructure integrity, and cell viability between four existing cryopreservation methods: liquid nitrogen (LN2) snap-freezing, LN2-cooled isopentane snap-freezing, RNAlater®-based freezing, and PCSF. RNA integrity was evaluated using agarose gel electrophoresis and RNA integrity number. Freezing-related microstructural damage in the muscle tissue was evaluated using ice crystal diameter and muscle fiber cross-sectional area. Protein integrity was evaluated using immunofluorescence staining. Cell viability was evaluated using trypan blue staining after primary muscle cell isolation. Results: PCSF preserved RNA integrity better than LN2 and isopentane, with a statistically significant difference. RNAlater preserved RNA integrity best. PCSF best controlled ice crystal size in myofibers, with a significant difference compared with LN2. The PCSF method best preserved the integrity of protein epitopes according to the mean fluorescence intensity results, with a significant difference. Cell viability was best preserved in the PCSF method compared with the other three methods, with a significant difference. Conclusion: PCSF protected the RNA integrity, microstructural integrity, protein integrity, and cell viability of skeletal muscle tissue. The application of PCSF in biobank practice is recommended as a multi-experiment-compatible cryopreservation method.

Genomics of fetal haemoglobin: a targeted approach for reticulocyte transcriptome study.

Background: Fetal haemoglobin (HbF) remains a major sickle cell disease modifier. The mechanism of HbF synthesis has been studied for several decades with the intention of increasing interventions for sickle cell disease (SCD), including drugs. However, the complex mechanism of HbF synthesis is influenced by multiple genetic factors interacting with environmental factors. In order to capture useful genetic information, especially with limited resources, one has to carefully design the study. This includes choosing the relevant participants, the correct phenotyping, the choice of samples, and the right genomic assays. This paper describes the approach undertaken as part of preparations for a reticulocyte transcriptome study intended to discover genes associated with HbF decline in newborns in Tanzania. Results: Of the 152 newborns enrolled in the larger study, 40 babies were selected for the reticulocyte transcriptome study based on their HbF levels at birth and later stage of life. Of these, 30 individuals were included under the category of high HbF levels ranging from 72.6-90% and the remaining 10 under the category of low HbF levels ranging from 5.9 - 10.3%. The reticulocyte enrichment recovery purity ranged from 85% - 97%. The total RNA concentrations obtained were >250 ng total RNA, with the average purity of 1.9 (A 260/280) respectively. The total concentration obtained was sufficient for the transcriptome and other downstream assays. Conclusion: We have documented important steps and factors to consider in identifying the relevant participants and required laboratory sample processes prior to the final stage, which involves total reticulocyte RNA sequencing.

The influence of tissue pH and RNA integrity number on gene expression of human postmortem brain.

Background: Evaluating and controlling confounders are necessary when investigating molecular pathogenesis using human postmortem brain tissue. Particularly, tissue pH and RNA integrity number (RIN) are valuable indicators for controlling confounders. However, the influences of these indicators on the expression of each gene in postmortem brain have not been fully investigated. Therefore, we aimed to assess these effects on gene expressions of human brain samples. Methods: We isolated total RNA from occipital lobes of 13 patients with schizophrenia and measured the RIN and tissue pH. Gene expression was analyzed and gene sets affected by tissue pH and RIN were identified. Moreover, we examined the functions of these genes by enrichment analysis and upstream regulator analysis. Results: We identified 2,043 genes (24.7%) whose expressions were highly correlated with pH; 3,004 genes (36.3%) whose expressions were highly correlated with RIN; and 1,293 genes (15.6%) whose expressions were highly correlated with both pH and RIN. Genes commonly affected by tissue pH and RIN were highly associated with energy production and the immune system. In addition, genes uniquely affected by tissue pH were highly associated with the cell cycle, whereas those uniquely affected by RIN were highly associated with RNA processing. Conclusion: The current study elucidated the influence of pH and RIN on gene expression profiling and identified gene sets whose expressions were affected by tissue pH or RIN. These findings would be helpful in the control of confounders for future postmortem brain studies.

High purity and integrity RNA from human cell lines stored in liquid nitrogen for over 20 years.

Biobanks are a key resource for obtaining human cell lines for biomedical research, including for drug development projects. Such projects often include comparative RNA-sequencing of large panels of human cell lines from individuals affected by certain disorders and healthy controls, or from individuals with different drug response phenotypes. RNA extractions are typically done from growing cell cultures, a process that may take several weeks. However, maintaining large numbers of cell lines in parallel increases the project workload. Here, we show that extracting RNAs directly from frozen vials of human cell lines stored for over 20 years in a liquid nitrogen freezer yields RNAs with the high purity and integrity parameters that conform to those required for optimal RNA-sequencing and are closely similar to those obtained for RNAs extracted from growing human cell lines.

Effects of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens: A systematic quantitative assessment.

Introduction: Blood samples are the most common biospecimen in biobanks, and RNA from such blood samples is an important material for biomedical research. High-quality RNA is essential for consistent, reliable results. Preanalytical environmental conditions can affect the quality of blood RNA. Here, we carried out a quantitative assessment of the influence of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens in biobanks. Methods: Before RNA purification, blood samples from volunteers were exposed to 4 °C for 2, 6, 12, 24, or 48 h, 3 days, or 1 week, or exposed to room temperature (22-30 °C) for 1, 2, 6, 12, or 24 h. Hemolyzed samples were collected from laboratory department and some of them were prepared using the freeze-thaw method. After exposure to different preanalytical environmental conditions, the RNA simple Total RNA Kit was used to purify the RNA, following which a NanoDrop™ One and Qsep 100 Bio-Fragment Analyzer were used to assess RNA concentration, purity, and integrity. In addition, a part of the RNA was immediately reverse transcribed into cDNA when it was purified, then the relative expression levels of 18S, ACTB, HIF1α, HMOX1, and MKI67 were determined by real-time quantitative PCR. Finally, 30 blood samples were collected from the surplus samples in our laboratory department to assess their RNA quality without knowledge of their storage conditions (duration/temperature). Results: For blood samples stored at 4 °C, there was a significant difference between the RNA integrity after 1 week compared to after 2 h. For blood samples stored at room temperature (22-30 °C), the RNA integrity was also significantly different at 6 h and 0 h. Hemolysis caused by freeze-thawing severely affected RNA quality, whereas clinical hemolysis generally produced no significant effects. Moreover, expression of 18S, ACTB, HIF1α, HMOX1, and MKI67 in whole blood stored under different conditions showed irregular changes, suggesting that preservation conditions are also important for gene expression. Conclusion: RNA integrity was qualified for blood samples stored at 4 °C for up to 72 h or at room temperature (22-30 °C) for up to 2 h. Hemolysis usually does not affect the RNA quality of blood samples unless the hemolysis method damages leukocytes.

An optimized TRIzol-based method for isolating RNA from adipose tissue.

High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Many studies have made efforts to optimize methods for isolating RNA from adipose tissue through combinations of column-based kits and phenol-chloroform methods, or through in-house protocols. However, the considerable complexity of these protocols and the various kits/materials required hamper their wide use. Herein, we describe an optimized protocol based on TRIzol reagent, which is the most accessible ready-to-use reagent for nucleic acid and/or protein isolation in laboratories. This article provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich specimens for downstream applications.

Impact of Sample Processing and Storage Conditions on RNA Quality of Fresh-Frozen Cancer Tissues.

Background: A biobank is a central resource that supports basic and clinical research. RNA quality of fresh-frozen tissue specimens in the biobank is highly associated with the success of downstream applications. Therefore, it is very important to evaluate the impact of tissue processing and storage conditions on RNA quality. Methods: A total of 238 surgically removed tissue specimens, including esophagus, lung, liver, stomach, colon, and rectal cancer, were used to evaluate RNA quality. Two tissue homogenization methods, manual and TissueLyser, were compared and the impacts of temperature fluctuation, tissue types, storage period, and clinicopathological parameters on RNA quality were analyzed. Results: RNA integrity was not influenced by tissue homogenization methods and tissue types. However, RNA integrity number (RIN) values were significantly correlated with temperature fluctuation. When the power of a -80°C freezer was cut off, RNA integrity of frozen tissues was not significantly affected until the temperature increased to 0°C. When the temperature rose to room temperature and remained for 4 hours, RNA integrity was almost completely destroyed. In addition, various cancer tissues with short-term storage at -80°C (<5 years) or high tumor differentiation had higher RINs. Conclusions: Tissue processing and storage conditions affected RNA quality of fresh-frozen cancer tissues. It is necessary to keep storage temperature stable and keep specimens at ultralow temperatures during homogenization. Also, for a biobank containing multiple types of cancer tissue samples, it is better to store them in liquid nitrogen if the storage duration is more than 5 years.

Areas of Crush Nuclear Streaming Should Be Included as Tumor Content in the Era of Molecular Diagnostics.

Degenerated tissues are frequently observed in malignant tumors, but are not analyzed. We investigated whether nuclear streaming and necrosis samples could be used for genetic analysis to expand the sample pool. A total of 81 samples were extracted from small cell carcinoma and lymphoma FFPE tissue blocks and classified into three histological cohorts: 33 materials with well-preserved tumor morphology, 31 nuclear streaming samples, and 17 necrosis samples. DNA and RNA integrity numbers, percentage of RNA fragments with >200 nucleotides, and next-generation sequencing quality metrics were compared among the cohorts. DNA quality did not significantly differ between nuclear streaming materials and materials with well-preserved morphology, whereas that of the necrosis samples was inferior. RNA quality decreased in the following order: materials with well-preserved morphology > nuclear streaming > necrosis. The sequencing metrics did not differ significantly between the nuclear streaming samples and materials with well-preserved morphology, and reliable variants were detected. The necrosis samples extracted from resections exhibited sequencing failure and showed significantly fewer on-target aligned reads and variants. However, variant allele frequency did not differ among the cohorts. We revelated that DNA in nuclear streaming samples, especially within biopsies, could be used for genetic analysis. Moreover, degenerated non-tumor cells should be counted when evaluating tumor content to avoid misinterpreting the variant allele frequency.

Isolation of High-Quality Total RNA from Small Animal Articular Cartilage for Next-Generation Sequencing.

Articular cartilage is characterized by a low density of chondrocytes surrounded by an abundant extracellular matrix (ECM) consisting of a dense mixture of collagens, proteoglycans, and glycosaminoglycans. Due to its low cellularity and high proteoglycan content, it is particularly challenging to extract high-quality total RNA suitable for sensitive high-throughput downstream applications such as RNA sequencing (RNA-Seq). Available protocols for high-quality RNA isolation from articular chondrocytes are inconsistent, resulting in suboptimal yield and compromised quality. This poses a significant difficulty in the application of RNA-Seq to study the cartilage transcriptome. Current protocols rely either on dissociation of cartilage ECM by collagenase digestion or pulverizing cartilage using various methods prior to RNA extraction. However, protocols for cartilage processing vary significantly depending on the species and source of cartilage within the body. Protocols for isolating RNA from human or large mammal (e.g., horse or cattle) cartilage samples are available, but this is not the case for chicken cartilage, despite the species being extensively used in cartilage research. Here, we present two improved RNA isolation protocols based on pulverization of fresh articular cartilage using a cryogenic mill or on enzymatic digestion using 1.2% (w/v) collagenase II. Our protocols optimize the collection and tissue processing steps to minimize RNA degradation and enhance RNA purity. Our results show that RNA purified from chicken articular cartilage using these methods has appropriate quality for RNA-Seq experiments. The procedure is applicable for RNA extraction from cartilage from other species such as dog, cat, sheep, and goat. The workflow for RNA-Seq analysis is also described here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of total RNA from pulverized chicken articular cartilage Alternate Protocol: Extraction of total RNA from collagen-digested articular cartilage Support Protocol: Dissection of chicken articular cartilage from the knee joint Basic Protocol 2: RNA sequencing of total RNA from chicken articular cartilage.

A Comparative Study of Three Nucleic Acid Integrity Assay Systems.

Background: The measurement of nucleic acid quality, especially the analysis of integrity, is a key step for many downstream experiments in biomedical research and quality control of biomaterials. General gel electrophoresis is a traditional method for nucleic acid integrity analysis. Currently, more electrophoresis techniques are becoming standardized and automated operations with higher precision. In this study, we have evaluated the comparability and bias of the outcomes from three commercial assay systems. Methods: Seventy-two deoxyribonucleic acid (DNA) and 67 ribonucleic acid (RNA) samples were selected for methodological comparison among different systems. The DNA Quality Number (DQN) and RNA Quality Number (RQN) of BIOptic Qsep400, DNA Quality Score (DQS) and RNA Quality Score (RQS) of PerkinElmer Labchip GX Touch HT were separately compared with the DNA Integrity Number (DIN) and RNA Integrity Number (RINe) of the Agilent 4200 TapeStation according to Clinical and Laboratory Standards Institute (CLSI) guideline (EP09-A3). Results: The biases of the mean estimated between DQN and DIN, DQS and DIN both exceeded the acceptance criteria. The Passing-Bablok regression analysis between DQN and DIN, and the Deming regression analysis between DQS and DIN, showed the biases were both within the acceptance criteria, and the bias between DQN and DIN was smaller. For the comparisons of RQN and RINe, RQS and RINe, the regression analyses revealed the biases were both within the acceptance criteria. The bias of the mean estimated between RQS and RINe was outside of the acceptance criteria. Conclusions: There was a good comparability in nucleic acid integrity detection between BIOptic Qsep400 and PerkinElmer Labchip GX Touch HT with the Agilent 4200 TapeStation. However, the bias and linear correlations require more attention between systems.

MAPKAPK2-centric transcriptome profiling reveals its major role in governing molecular crosstalk of IGFBP2, MUC4, and PRKAR2B during HNSCC pathogenesis.

Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.