Table of Contents
What is RiboGreen assay?
RiboGreen® RNA Quantitation Reagent is an ultra- sensitive fluorescent nucleic acid stain. It is a simple and rapid procedure for measuring RNA concentration in solution for several molecular biology procedures.
What does RiboGreen bind to?
DNA
RiboGreen® reagent also binds to DNA. Fluorescence in samples that is due to RiboGreen® reagent binding to DNA can be eliminated by pre-treating the sample with RNase-free DNase, ensuring that the entire sample fluorescence is due to dye bound to RNA.
What is OliGreen?
Quant-iT™ OliGreen® oligonucleotide reagent is an ultra-sensitive fluorescent nucleic acid stain. Use of OliGreen allows performance of simple and rapid procedures for quantitating oligonucleotides and single-stranded DNA (ssDNA).
How do you quantify RNA?
The traditional method for assessing RNA concentration and purity is UV spectroscopy. The absorbance of a diluted RNA sample is measured at 260 and 280 nm. The nucleic acid concentration is calculated using the Beer-Lambert law, which predicts a linear change in absorbance with concentration (Figure 1).
How do you quantify single stranded DNA?
For the most accurate DNA quantitation, samples should be quantitated first with the QuantiFluor® dsDNA System to specifically measure dsDNA, treated with shrimp DNase and then quantitated using the QuantiFluor™ ssDNA System to specifically measure ssDNA.
What is a good RIN value?
It provides a numerical score (range 1–10) for RNA quality. A higher RIN value indicates a higher degree of RNA integrity [20]. It is generally accepted that for samples with high inter granularity, the RIN level should be about 7–8.
How is RNA quality measured?
The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret.
Can you PCR single-stranded DNA?
Single-stranded DNA can be generated by conventional asymmetric or real-time asymmetric PCR (9,10).
Are primers single or double-stranded?
single-stranded
A primer, as related to genomics, is a short single-stranded DNA fragment used in certain laboratory techniques, such as the polymerase chain reaction (PCR).
What does RIN measure?
RIN is a standard method of measuring RNA integrity and can be used to evaluate the quality of RNA produced by new RNA isolation techniques.
How is RIN value calculated?
RIN is determined by the result of microcapillary electrophoretic RNA separation. Several features, including the total RNA ratio, the height of the 28S peak, the fast area ratio, and the marker height, are taken into consideration in the RIN algorithm.
What is a good RNA quality?
A 28S/18S ratio of two is considered to be good quality RNA. Traditional separation of total RNA on denaturing agarose gel electrophoresis followed by ethidium bromide staining. The 28S/18S RNA bands in this figure have an intensity ratio around 2 that would be considered good to very good quality RNA.
Are primers single or double stranded?
Does PCR produce ssDNA or dsDNA?
Liner-After-The-Exponential (LATE]-PCR is a novel, highly robust asymmetric PCR method that uses unequal concentrations of primers to generate large amounts of single-stranded DNA (ssDNA) together with a small fixed amount of double-stranded DNA (dsDNA) [29,30].
Are primers DNA or RNA?
A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. In living organisms, primers are short strands of RNA. A primer must be synthesized by an enzyme called primase, which is a type of RNA polymerase, before DNA replication can occur.
What does a RIN number tell you?
Index. The RNA Integrity Number (RIN) The RNA integrity number (RIN) is a tool designed to help scientists estimate the integrity of total RNA samples. The RIN extension automatically assigns an integrity number to a eukaryote total RNA sample analyzed on the Agilent 2100 bioanalyzer.
What is RiboGreen RNA reagent?
These RNA quantification kits contain Quant-iT RiboGreen RNA reagent, which is one of the most sensitive detection dyes for the quantitation of RNA in solution, with linear fluorescence detection in the range of 1–200 ng of RNA.
What is in the Quant-iT RiboGreen RNA assay kit?
Description The Quant-iT™ RiboGreen® RNA Assay Kit contains Quant-iT™ RiboGreen® RNA reagent as well as buffers and RNA standards. Quant-iT™ RiboGreen® RNA reagent is one of the most sensitive detection dyes for the quantitation of RNA in solution, with linear fluorescence detection in the range of 1-200 ng of RNA.
What is RiboGreen dye used for?
( February 2009) RiboGreen is a proprietary fluorescent dye that is used in the detection and quantification of nucleic acids, including both RNA and DNA. It is synthesized and marketed by Molecular Probes / Invitrogen (a division of Life Technologies, now part of Thermo Fisher Scientific) of Eugene, Oregon, United States.
Where is RiboGreen made?
It is synthesized and marketed by Molecular Probes / Invitrogen (a division of Life Technologies, now part of Thermo Fisher Scientific) of Eugene, Oregon, United States. In its free form, RiboGreen exhibits little fluorescence and possesses a negligible absorbance signature.