Quantification of RNA

Knight JC, Keating BJ, Rockett KA, Kwiatkowski DP. In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nat Genet 2003 33 469-475. 

Nkengasong JN, Bile C, Kalou M, Maurice C, Boating E, Sasson-Moroko M> et al. Quantification of RNA in HIV Type 1 subtypes D and G by NucliSens and Amplicor Assays in Abidjan, Ivory Coast. AIDS Res Hum Retroviruses 1999 15 495-8. 

The first attempts to enable gene expression analysis by MS were reported only recently, when Smith and coworkers appHed the Invader assay (see Section 5.4.1.5 and Figure 5.12) to the detection and relative quantification of RNA [224]. In order to allow an inter-spectrum (and thus inter-sample) correlation. Invader assays of multiple target genes were multiplexed and a reference gene was included for normalization of the data. 

Several methods are available to monitor transcription levels of tens of thousands of genes rapidly and simultaneously. The quantification of RNA species by sequence-specific annealing (hybridization) to complementary DNA probes arrayed on a substrate (microarray) was developed by Schena etal. (1995). Sample RNA was submitted to reverse transcription and fluorescent labeling. Thereby, a quantitative parameter was produced that could be measured as a localized signal after hybridization to the arrayed sensors. Comparison by competitive hybridization of two RNA samples labeled with two different dyes (Cy3 and Cy5) resulted in expression ratios of the two sources (e.g., of tumor and nontumor tissue). Whereas sensors were originally taken from libraries of DNA clones (cDNA), present-day microar-... 

Chateigner-Boutin, A. Small, I. A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons. Nucleic Acids Res. 2007, 35, el 14/1-el 14/8. 

Morozkin, E. S. Laktionov, P. R Rykova, E. Y Vlassov, V. V. Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids. Anal. Biochem. 2003,322,48-50. 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

A prototype bDNA assay was developed for quantification of HGV/GBV-C RNA in serum (Pessoa et al, 1997). The assay employed target probes based on the relatively conserved sequence in the 5 untranslated region of the HGV/GB V-C genome. Preamplifier molecules and incorporation of isoC and isoG into the sequences common to bDNA assays were used to enhance the analytical sensitivity. The provisional limit of detection was 32,500 genome equivalents/ml based on dilutions of a 700-nucleotide synthetic HGV/GBV-C RNA transcript. The run-to-run variance of the assay was <15%. 

Detmer, J et al. (1996). Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology. J. Clin. Microbiol. 34,901-907. 

Pachl, C., el al. (1995). Rapid and precise quantification of HTV-1 RNA in plasma using a branched DNA signal amplification assay. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 8,446-454. 

Revets, H., etal. (1996). Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HTV Monitor, and QUAN l lPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol 34,1058-1064. 

The chemical modification of nucleic acids at specific sites within individual nucleotides or within oligonucleotides allows various labels to be incorporated into DNA or RNA probes. This labeling process can produce conjugates having sensitive detection properties for the localization or quantification of oligo binding to a complementary strand using hybridization assays. 

Quantitative RT PCR (qRT PCR) can be used to accurately determine the levels of messages within given preparations of RNA. qRT PCR thermocyclers provide rapid online detection and quantification of mRNA, however, the initial purchase cost and the cost of reagents may be prohibitive for some laboratories. Methods of semiquantitative RT PCR have been used and good descriptions of these techniques are available (Samhrook and Russell, 2001). However, the same cDNA populations should not be used for differential display reactions and verification that a potential differential display band represents a differentially expressed gene. For this reason, independent cDNA samples should be prepared if both the screening and verification methods rely on PCR. qRT PCR, therefore, should be used in conjunction with other methods to verify that a differential display band represents a differentially expressed gene. 

HCV and HIV-1). The bDNA assay is being much employed for the quantification of messenger RNA. Moreover, for the detection of viral and pathogenic disorders based on alkahne-phosphatase-sensitive dioxetanes, several assay methods are available these include the Polymerase-Chain-Reaction (PCR) amphfication, probe ligation, strand-displacement amplification and the ligase chain reaction. ... 

This procedure has been developed for quantification of the three types of macromolecules in tissue extracts, where other hiomolecules are also present. Small dissolved amounts of DNA, RNA, or protein, especially when no material should be consumed and no interfering substances are in the solution, maybe estimated by UV photometry, but a discrimination between DNA and RNA is impossible by reading absorbencies (cf. Protocol 1.2.5). 

Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., and Altman, R. B. (2005). SAFA Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344—354. 

Recent simulation work (Chen et al., 2009) focused on comparative quantification of the accumulation of different types of group I monovalent cations around the Tar—Tar kissing loop. The analysis identified three salient features regarding the distributions of accumulated ions around Tar—Tar (1) The ionic atmosphere can be quantified in terms of free energy layers around the RNA using the methods described in detail below. The most favorable free energy levels (AG < —1.5 kcal/mol) are occupied... 

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