Lowering the quantification limit of the QubitTM RNA HS Assay using RNA spike-in (original) (raw)
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Analytical Biochemistry, 2009
Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng ll À1 range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-ll sample in the 500 to 5-ng ll À1 range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng ll À1 range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng ll À1 range. However, the Agilent kits require 1 ll of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.
A Comparative Analysis of Three Methods Used for Rna Quantitation
2015
The high RNA sample quality is essential for downstream molecular biology applications. Two simultaneous conditions should be accomplished by the RNA samples: the structural integrity of the molecules and an adequate concentration. The objective of this study is to do a comparative analysis between three different methods of measuring RNA concentration. The three methods considered here are: UV spectrophotometry, spectrophotofluorimetry, and microfluidic capillary electrophoresis. Sixteen RNA samples were assayed by these three methods. The principles and the results of each method as well as advantages, disadvantages and perturbations factors are analyzed and discussed. Fluorescent labelling of RNA gives more accurate results even in the presence of the frequent contaminants like DNA and proteins. Knowing the strength and the limits of each method required for RNA quantitation, the scientist can choose the most cost-effective protocol.
2021
The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors’ plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by comprom...
Challenges for Accurate Quantification of RNA
Reviews in Agricultural Science, 2021
Ribonucleic acid (RNA) quantification is an essential technique in biology. There has been remarkable progress in RNA quantification techniques over the recent years; however, the specificity of these techniques to quantify a very small amount of RNA is doubtful because of factors which can inhibit precise quantification. To develop a technique that leads to the most sensitive RNA quantification, these problems must be overcome. In this article, we first review the factors that inhibit precise quantification of RNA: the quality of RNA, secondary structure of RNA, efficiency of the enzyme reaction, annealing conditions, limitations of the experimental protocol and equipment, and detection sensitivity of the equipment. Next, we discuss the possible methods which contribute to these factors: RNA quality control focused on target RNA degradation, isothermal amplification, techniques for avoiding amplification errors, RNase H-dependent PCR, targeting using a fluorescent-labeled probe, targeting using a padlock probe, bridged/locked nucleic acid (BNA/LNA) and peptide nucleic acid (PNA), and the clustered regularly interspaced short palindromic repeat (CRISPR) system. One of the goals for the development of an ultrasensitive RNA quantification technique is the absolute quantification of RNA. Here, we discuss the techniques used for this type of RNA quantification.
2021
The use of blood-based extracellular RNA (cell-free RNA; exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing, and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated ten blood collection tubes, three time intervals between blood draw and downstream processing, and eight RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics on deep transcriptome profiling of both small and messenger RNA from healthy donors’ plasma or serum was assessed for each pre-analytical variable separately and for interactions between a selected set of pre-analytics, resulting in 456 extracellular transcriptomes. Making use of 189 synthetic spike-in RNAs, the processing and analysis workflow was controlled. When comparing blood collection tubes, so-called prese...
A high-throughput protocol for message RNA quantification using RNA dot-blots
Analytical Biochemistry, 2014
This study develops a method to rapidly measure the relative abundance of mRNA in total RNA samples using a dot-blotting technique and biotin-labeled detection probes that recognize the polyadenylate tail on mRNA. We demonstrate the effectiveness of this technique by determining the relative total amounts of mRNA in three tissues of turtles (Trachemys scripta elegans) exposed to normoxic versus anoxic conditions. The data emphasize the usefulness of the method for the simple and rapid analysis of relative total mRNA levels for a variety of comparison purposes.
Science China Life Sciences, 2013
RNA-Seq promises to be used in clinical settings as a gene-expression profiling tool; however, questions about its variability and biases remain and need to be addressed. Thus, RNA controls with known concentrations and sequence identities originally developed by the External RNA Control Consortium (ERCC) for microarray and qPCR platforms have recently been proposed for RNA-Seq platforms, but only with a limited number of samples. In this study, we report our analysis of RNA-Seq data from 92 ERCC controls spiked in a diverse collection of 447 RNA samples from eight ongoing studies involving five species (human, rat, mouse, chicken, and Schistosoma japonicum) and two mRNA enrichment protocols, i.e., poly(A) and RiboZero. The entire collection of datasets consisted of 15650143175 short sequence reads, 131603796 (i.e., 0.84%) of which were mapped to the 92 ERCC references. The overall ERCC mapping ratio of 0.84% is close to the expected value of 1.0% when assuming a 2.0% mRNA fraction in total RNA, but showed a difference of 2.8-fold across studies and 4.3-fold among samples from the same study with one tissue type. This level of fluctuation may prevent the ERCC controls from being used for cross-sample normalization in RNA-Seq. Furthermore, we observed striking biases of quantification between poly(A) and Ri-boZero which are transcript-specific. For example, ERCC-00116 showed a 7.3-fold under-enrichment in poly(A) compared to RiboZero. Extra care is needed in integrative analysis of multiple datasets and technical artifacts of protocol differences should not be taken as true biological findings.
Inter-Laboratory Variability in Array-Based RNA Quantification Methods
Genomics Insights, 2013
Ribonucleic acids (RNA) are hypothesized to have preceded their derivatives, deoxyribonucleic acids (DNA), as the molecular media of genetic information when life emerged on earth. Molecular biologists are accustomed to the dramatic effects a subtle variation in the ribose moiety composition between RNA and DNA can have on the stability of these molecules. While DNA is very stable after extraction from biological samples and subsequent treatment, RNA is notoriously labile. The short half-life property, inherent to RNA, benefits cells that do not need to express their entire repertoire of proteins. The cellular machinery turns off the production of a given protein by shutting down the transcription of its cognate coding gene and by either actively degrading the remaining mRNA or allowing it to decay on its own. The steady-state level of each mRNA in a given cell varies continuously and is specified by changing kinetics of synthesis and degradation. Because it is technically possible to simultaneously measure thousands of nucleic acid molecules, these quantities have been studied by the life sciences community to investigate a range of biological problems. Since the RNA abundance can change according to a wide range of perturbations, this makes it the molecule of choice for exploring biological systems; its instability, on the other hand, could be an underestimated source of technical variability. We found that a large fraction of the RNA abundance originally present in the biological system prior to extraction was masked by the RNA labeling and measurement procedure. The method used to extract RNA molecules from cells and to label them prior to hybridization operations on DNA arrays affects the original distribution of RNA. Only if RNA measurements are performed according to the same procedure can biological information be inferred from the assay read out.
BMC Biotechnology, 2014
Background: This study compared the performance of five commercially available kits in extracting total RNA from small eukaryotic tissue samples (<15 mg). Total RNA was isolated from fathead minnow (Pimephales promelas) tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA HT kits. Kit performance was evaluated via measures of RNA quantity (e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260 and 280 nm, RNA integrity number (RIN), presence of gDNA). Results: With the exception of embryos, each kit generally extracted ≥5 μg of total RNA from each sample. With regard to RNA quality, the RINs of RNA samples isolated via the Plus Mini and Maxwell® 16 kits were consistently higher than those of samples extracted via the remaining three kits and for all tissues, these kits produced intact RNA with average RIN values ≥7. The Plus Universal and SimplyRNA HT kits produced moderately degraded (RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit tended to exhibit a high degree of degradation (RIN values <5).