Impact of RNA degradation on next-generation sequencing transcriptome data.

RNA sequencing is an innovative technology to study transcriptomes in both biological and clinical research. However, clinical specimens from patients undergoing surgical operations have a major challenge due to sample degradation. This study replicated the process of RNA degradation by maintaining cells at room temperature to achieve none, slight, middle, and high levels of RNA degradation with decreasing RNA integrity numbers (RIN) of approximately 9.8, 6.7, 4.4, and 2.5, respectively. Next, the differential expression of mRNA and long non-coding RNA (lncRNA) was analyzed in the four degradation groups along with pathway enrichment analysis. The results showed that the similarity of lncRNAs exhibited significant differences even for a slight level of RNA degradation compared with the non-degraded RNA sample. Also, the RNA degradation process was found to be universal, global, and random; the differentially expressed genes increased with an increase in degradation but the pathway enrichment phenomenon was not significantly observed.

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.

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.

Host Poly(A) Polymerases PAPD5 and PAPD7 Provide Two Layers of Protection That Ensure the Integrity and Stability of Hepatitis B Virus RNA.

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize hepatitis B virus (HBV) RNA via the interaction with the viral posttranscriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, hepatitis B surface antigen (HBsAg) production, and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies; therefore, it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (50% effective concentration [EC50] ranged from 1.4 to 6.8 nM) and in vivo by 0.94 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity toward the inhibitory effect of AB-452. Although neither single knockout (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO did not result in any measurable changes within the HBV poly(A) tails, but cells with both PAPD5 and PAPD7 KO show reduced HBsAg production and conferred complete resistance to AB-452 treatment. Our results indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5-targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication. IMPORTANCE Chronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence, and as such, therapies that aim at reducing HBsAg through destabilizing or degrading HBV RNA have been extensively investigated. Besides directly degrading HBV transcripts through antisense oligonucleotides or RNA silencing technologies, small-molecule compounds targeting host factors such as the noncanonical poly(A) polymerase PAPD5 and PAPD7 have been reported to interfere with HBV RNA metabolism. Herein, our antiviral and genetic studies using relevant HBV infection and replication models further characterize the interplays between the cis element within the viral sequence and the trans elements from the host factors. PAPD5/7-targeting inhibitors, with oral bioavailability, thus represent an opportunity to reduce HBsAg through destabilizing HBV RNA.

How long does the mRNA remains stable in untreated whole bovine blood?

BACKGROUND: High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. METHODS AND RESULTS: In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4 °C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage. CONCLUSION: Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.

Development of a 3':5' digital PCR assay to determine horse mRNA integrity.

Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3':5' assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3':5' assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysis of the mRNA integrity, but also of its quantity.

Yield and Integrity of RNA from Brain Samples are Largely Unaffected by Pre-analytical Procedures.

Gene expression studies are reported to be influenced by pre-analytical factors that can compromise RNA yield and integrity, which in turn may confound the experimental findings. Here we investigate the impact of four pre-analytical factors on brain-derived RNA: time-before-collection, tissue specimen size, tissue collection method, and RNA isolation method. We report no significant differences in RNA yield or integrity between 20 mg and 60 mg tissue samples collected in either liquid nitrogen or the RNAlater stabilizing solution. Isolation of RNA employing the TRIzol reagent resulted in a higher yield compared to isolation via the QIAcube kit while the latter resulted in RNA of slightly better integrity. Keeping brain tissue samples at room temperature for up to 160 min prior to collection and isolation of RNA resulted in no significant difference in yield or integrity. Our findings have significant practical and financial consequences for clinical genomic departments and other laboratory settings performing large-scale routine RNA expression analysis of brain samples.

Molecular analysis of H&E- and Papanicolau-stained samples-systematic review.

Molecular pathology allows the identification of causative agents in infectious diseases and detection of biomarkers important for prediction of disease susceptibility, diagnosis and personalized therapy. Accordingly, nucleic acid-based methods have gained a special role in clinical laboratories particularly to evaluate solid and hematological tumors. Extraction of nucleic acids is commonly performed in microdissected formalin-fixed paraffin-embedded (FFPE) or cytological samples that had been previously evaluated through the use of hematoxylin and eosin (H&E) or Papanicolau (Pap) stains, respectively. Although the effect of both stains on nucleic acids integrity has been explored by several authors, the results are not consistent and require further examination. Accordingly, the goal of this review was to assess the influence of H&E and Pap stains on DNA and RNA integrity and to address the mechanism by which each staining compromises molecular based-analysis. The analyzed studies demonstrate that H&E- and Pap-staining result in low DNA recovery and some degree of DNA fragmentation. Additionally, it is concluded that hemalum inhibits PCR by interfering with DNA extraction, preventing DNA polymerase attachment and possibly by rescuing divalent cations. Accordingly, proper sample purification and adjustment of PCR conditions are of key importance to achieve satisfactory results by PCR in H&E- and Pap-stained samples. Furthermore, although H&E results in RNA fragmentation, it is possible to perform expression analysis in H&E-stained frozen sections, using RNase-free conditions, low amounts of hematoxylin and a rapid protocol from sample collection to RNA analysis. It The effect of Pap-staining on RNA integrity remains to be determined.

Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity.

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3'-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Laser-capture microdissection of murine lung for differential cellular RNA analysis.

The lung tissue contains a heterogeneous milieu of bronchioles, epithelial, airway smooth muscle (ASM), alveolar, and immune cell types. Healthy bronchiole comprises epithelial cells surrounded by ASM cells and helps in normal respiration. In contrast, airway remodeling, or plasticity, increases surrounding of bronchial epithelium during inflammation, especially in asthmatic condition. Given the profound functional difference between ASM, epithelial, and other cell types in the lung, it is imperative to separate and isolate different cell types of lungs for genomics, proteomics, and molecular analysis, which will improve the diagnostic and therapeutic approach to treat cell-specific lung disorders. Laser capture microdissection (LCM) is the technique generally used for the isolation of specific cell populations under direct visual inspection, which plays a crucial role to evaluate cell-specific effect in clinical and preclinical setup. However, maintenance of tissue RNA quality and integrity in LCM studies are very challenging tasks. It is obvious to believe that the major factor affecting the RNA quality is tissue-fixation method. The prime focus of this study was to address the RNA quality factors within the lung tissue using the different solvent system to fix tissue sample to obtain high-quality RNA. Paraformaldehyde and Carnoy's solutions were used for fixing the lung tissue and compared RNA integrity in LCM captured lung tissue samples. To further confirm the quality of RNA, we measured cellular marker genes in collected lung tissue samples from control and mixed allergen (MA)-induced asthmatic mouse model using qRT-PCR technique. RNA integrity number showed a significantly better quality of RNA in lung tissue samples fixed with Carnoy's solution compared to paraformaldehyde solution. Isolated RNA from MA-induced asthmatic murine lung epithelium, smooth muscle, and granulomatous foci using LCM showed a significant increase in remodeling gene expression compared to control which confirm the quality and integrity of isolated RNA. Overall, the study concludes tissue fixation solvent can alter the quality of RNA in the lung and the outcome of the results.