Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity
TL;DR: RNA A260/280 ratios are found to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 and the ability to detect protein contamination was significantly improved when RNA wasSpectrophotometrically analyzed in an alkaline solution.
Abstract: The ratio of absorbance at 260 and 280 nm (the A260/280 ratio) is frequently used to assess the purity of RNA and DNA preparations. Data presented in this report demonstrate significant variability in the RNA A260/280 ratio when different sources of water were used to perform the spectrophotometric determinations. Adjusting the pH of water used for spectrophotometric analysis from approximately 5.4 to a slightly alkaline pH of 7.5-8.5 significantly increased RNA A260/280 ratios from approximately 1.5 to 2.0. Our studies revealed that changes in both the pH and ionic strength of the spectrophotometric solution influenced the A260/280 ratios. In addition, the ability to detect protein contamination was significantly improved when RNA was spectrophotometrically analyzed in an alkaline solution. UV spectral scans showed that the 260-nm RNA absorbance maximum observed in water was shifted by 2 nm to a lower wavelength when determinations were carried out in Na2HPO4 buffer at a pH of 8.5. We found RNA A260/280 ratios to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 in 1-3 mM Na2HPO4 buffer.
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TL;DR: The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.
Abstract: Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.
1,741 citations
TL;DR: This review addresses the mathematics of RT-PCR, choice of RNA standards, and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification) and practical considerations in experimental design.
Abstract: Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.
1,217 citations
TL;DR: Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols, enabling the assessment of nucleic Acid concentrations ranging from 1 pg/ μL to 15,000 ng/μL with minimal consumption of sample.
Abstract: Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.
355 citations
TL;DR: The cytoplasmic domain of LRP6 is not similar to other members of the LDLR family, while comparison with LRP5 reveals proline-rich motifs that may mediate protein-protein interactions, likely to comprise a new class of the HDLR family.
224 citations
TL;DR: In this article, the authors discuss methods to measure single-cell mass and their relative strengths and weaknesses for different applications, and propose a method to measure the mass of a single cell.
Abstract: This Perspective discusses methods to measure single-cell mass and their relative strengths and weaknesses for different applications.
218 citations
References
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TL;DR: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described, providing a pure preparation of undegraded RNA in high yield and can be completed within 4 h.
65,881 citations
Journal Article•
TL;DR: The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
Abstract: This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
2,807 citations
TL;DR: Myoblast and myotube nuclei display similar patterns of heterogeneous acetylcholine receptor subunit mRNA expression, and Simultaneous localization of neurofilament protein, nuclear DNA and acetylCholine receptor mRNA.
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