Quantification Of Ribonucleic Acid Research Articles

Privacy preserving validation for multiomic prediction models

Reproducibility of results obtained using ribonucleic acid (RNA) data across labs remains a major hurdle in cancer research. Often, molecular predictors trained on one dataset cannot be applied to another due to differences in RNA library preparation and quantification, which inhibits the validation of predictors across labs. While current RNA correction algorithms reduce these differences, they require simultaneous access to patient-level data from all datasets, which necessitates the sharing of training data for predictors when sharing predictors. Here, we describe SpinAdapt, an unsupervised RNA correction algorithm that enables the transfer of molecular models without requiring access to patient-level data. It computes data corrections only via aggregate statistics of each dataset, thereby maintaining patient data privacy. Despite an inherent trade-off between privacy and performance, SpinAdapt outperforms current correction methods, like Seurat and ComBat, on publicly available cancer studies, including TCGA and ICGC. Furthermore, SpinAdapt can correct new samples, thereby enabling unbiased evaluation on validation cohorts. We expect this novel correction paradigm to enhance research reproducibility and to preserve patient privacy.

Effect of Sodium Tungstate on Nucleic Acid Extraction from Anaerobic Digestion Sludge —Efficacy of Fluorescence Measurement in the Ribonucleic Acid Quantification—

Effect of Sodium Tungstate on Nucleic Acid Extraction from Anaerobic Digestion Sludge —Efficacy of Fluorescence Measurement in the Ribonucleic Acid Quantification—

Precise RNA Quantification by Counting Individual RNA Molecules Using High-Sensitivity Capillary Flow Cytometry.

Precise determination of ribonucleic acid (RNA) concentration without the need for calibration was pursued by sequence-specific counting of individual RNA molecules. This approach eliminates the reverse transcription (RT) step required for polymerase chain reaction (PCR)-based quantification, which may hamper accurate measurements owing to uncertain yields of RT reactions. Target RNAs were tagged with a number of fluorescent oligonucleotide probes with complementary sequences. Tagged RNAs were exhaustively counted one by one using a high-sensitivity capillary-based flow cytometric setup. MS2 viral RNA was quantified as a model RNA for which essential parameters, including probe numbers, probe concentration, and hybridization conditions, were optimized for the best performance. Using 70 oligonucleotide probes, MS2 RNA was quantified with 2.0% relative standard deviation, and its validity was assessed by comparison with other RNA quantification methods such as droplet digital PCR and UV spectrophotometry. The observed comparability indicated that the proposed method is unlikely to have a substantial bias. It works for a substantially lower-level RNA than UV and avoids the potential variability in the yield of the RT reaction of RT-qPCR. Therefore, the proposed method could be a valuable addition to current methods and could be further developed as a standard reference method for RNA quantification.

8 Assessment of consistency in quantification of ribonucleic acid across multiple methods

Abstract Quantification and assessment of quality of ribonucleic acid (RNA) is essential for assays determining gene expression including qRT-PCR, northern blotting, and RNA sequencing. Methods assessing RNA abundance and quality vary in detection range and specificity, in part due to their differences in how quantification is achieved. Agilent chip technology utilizes fluorescent labeling of nucleic acids coupled to electrophoresis to separate RNA based on size. Nanodrop utilizes spectrophotometry assesses quantity nucleic acids based on absorption of light and allows for an estimation of purity using the A260/280 (nucleic acid to protein) ratio. RiboGreen assays utilize a fluorescent intercalating dye that specifically targets single-stranded RNA. The objective of this study was to determine the most accurate method of RNA quantification between these three assays. Male pituitary samples (n = 220) were collected at a local abattoir, anterior lobe isolated, flash frozen in methanol dry-ice bath, and stored at -80°C until further processing. Total RNA was isolated from all samples and samples were subjected to spectrophotometry, Agilent nano chip assay, and Ribogreen assay. Data were analyzed for differences in standard deviation of total yield (mg/g) across all methods and each pair of methods using an O’Brien test for unequal variances. Standard deviation differed due to quantification method (P = 0.0002). Over 5 times greater variability in quantification was observed between Agilent chip technology compared to nanodrop (P = 0.0036), 2 times greater between nanodrop and Ribogreen (P < 0.0001), and 10 times greater variation between Agilent and Ribogreen methods (P = 0.0029). The Ribogreen assay provides the smallest variation in quantification from sample to sample and is the most reliable method out of the three technologies for quantification of RNA; however, it does not address issues of RNA quality.

Circulating CD30+CD4+ T Cells Increase Before Human Immunodeficiency Virus Rebound After Analytical Antiretroviral Treatment Interruption.

Identification of nonviral markers of human immunodeficiency virus (HIV) infection that increase before viral rebound during analytical treatment interruption (ATI) may affect HIV persistence research. We previously showed that HIV ribonucleic acid (RNA) is enriched in CD30+CD4+ T cells in many individuals. Here, we studied CD30+CD4+ T-cell dynamics before ATI, during ATI (before detectable plasma RNA), and after HIV rebound. Peripheral blood mononuclear cells from 23 participants collected longitudinally from 5 Adult AIDS Clinical Trials Group studies incorporating ATI were included in this study. Flow cytometric characterization of expression of CD30 and markers of T-cell activation and exhaustion were performed along with HIV-1 RNA and deoxyribonucleic acid quantification and measurement of soluble plasma CD30 and CD30 ligand. The percentage of CD4+ T cells expressing CD30 significantly increased from pre-ATI to postinterruption time points before detectible viremia (1.65 mean relative increase, P = .005). Seventy-seven percent of participants experienced an increase in CD30+ cells before viral rebound. In contrast, there were no significant differences between pre-ATI and postinterruption pre-rebound time points in percentages of lymphocytes expressing CD69, CD38/HLA-DR, or PD-1 until after HIV recrudescence. CD30 may be a surrogate marker of early replication or viral transcriptional activity before detection by routine peripheral blood sampling.

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

Activation of Split RNA Aptamers: Experiments Demonstrating the Enzymatic Synthesis of Short RNAs and Their Assembly As Observed by Fluorescent Response

With the advancement of ribonucleic acid (RNA) research in the field of biochemistry, ensuring that undergraduate students have both the theoretical and practical knowledge of current, cutting-edge experimental techniques is of marked importance. Many current biochemistry experiments demonstrate various basic molecular biological techniques including isolation and quantification of nucleic acids, protein gel electrophoresis, and DNA amplification reactions. While covering a wide base of skills, there is an underutilization of RNA as a pedagogical tool. This multistep laboratory experiment introduces students to the emerging field of RNA nanotechnology and to the RNA assembly and subsequent conditional activations of preprogrammed functionalities such as fluorescence. To achieve this, a sequence of standard molecular techniques such as PCR, DNA purification, RNA transcription and purification, RNA quantification, and self-assembly followed by analysis with electrophoretic mobility shift assays and spectros...

Comparison of three commercial assays for the quantification of HIV-1 RNA in plasma from individuals infected with different HIV-1 subtypes

Commercial human immunodeficiency virus 1 (HIV-1) ribonucleic acid (RNA) quantification assays vary in their ability to quantify different subtypes of HIV-1, a problem in regions where multipte HIV-1 subtypes may be circulating. To assess commercial HIV-1 RNA quantification assays on two plasma panels. Panel 1 consisted of HIV-1 seronegative plasma 'spiked' with a known amount of cultured virus of different subtypes, and Panel 2 comprised plasma collected from individuals infected with different HIV-1 subtypes. The comparison involved the Amplicor HIV-1 reverse transcriptase-polymerase chain reaction (RT-PCR), Quantiplex branched DNA, and NucliSens HIV-1 QT assays. Panel 1 consisted of 11 plasma 'spiked' with cultured viruses of HIV-1 subtypes A-F, and Panel 2 included 33 plasma samples from 16 patients infected with subtypes A, B, C, E and G. In Panel 1, the Quantiplex branched deoxyribonucleic acid (bDNA) assay quantified subtypes A-F efficiently, comparable to published results from two other laboratories. The Amplicor RT-PCR assay quantified subtypes B, C, and D but was relatively less efficient with subtypes E, F, and did not or poorly quantified subtype A. Testing of Panel 2 showed some inter-assay differences. In contrast to Panel 1, the Amplicor RT-PCR assay performed variably with subtype A when compared with the Quantiplex bDNA and NucliSens QT assays, and higher viral load levels were generated with subtype E using the Amplicor RT-PCR assay. Subtypes B and C showed some inter-patient differences but the Quantiplex bDNA generally gave a lower quantification than the Amplicor RT-PCR and NucliSens QT assays. These studies confirm that commercial HIV-1 load assays vary in their ability to quantify different HIV-1 subtypes. This may be more apparent with individual patient samples than with 'spiked' panels. This variability emphasizes that it is preferable for patient samples to be tested with the same assay, and care should be taken where infection with unusual subtypes is suspected.

Biologic activity and quantification of messenger RNA coding for human chorionic somatomammotropin in normal and intrauterine growth-retarded pregnancies

Total ribonucleic acid (RNA) from human placentas obtained from normal and intrauterine growth-retarded (IUGR) pregnancies was translated in a reticulocyte cell-free system. Synthesis of human chorionic somatomammotropin (hCS) was estimated as a ratio of specific immunoprecipitated protein over total newly synthesized proteins. There is no significant difference between in vitro hCS synthesis directed by placental RNA from normal and IUGR pregnancies. Measurements of messenger RNA sequences coding for hCS, with a hCS complementary DNA probe, indicated that the hCS messenger RNA (mRNA) concentrations were similar for both groups. Low plasma hCS levels in pregnancies associated with growth-retarded fetuses can be explained by their significantly lower placental weights which correlate with their total RNA content. The total capacity of in vitro hCS production per placenta is significantly lower in this type of abnormal pregnancy. There is a good parallelism between the amount of hCS mRNA, its biologic activity tested in a cell-free system, and the secretion of hCS in the maternal circulation. These data suggest that there is no basic intracellular disturbance in hCS synthesis in placentas from fetal growth-retarded pregnancies.