Rna Detection Methods

A. Techniques Used for Detection of the RNA Product of In Vitro Transcription Reactions or RNA Isolated from Cells

Traditional RNA detection methods involve ethidium bromide staining following gel electrophoresis, RNA (Northern) blotting techniques, RNase protection, and direct radiolabeling of RNA [11]. None of these traditional methods for RNA detection are suitable for automation and HTS. More recently, a number of novel methods have been developed to detect small amounts of specific RNA. The most commonly used methods involve the polymerase chain reaction (PCR) [12,13] and include amplification of the specific RNA product and subsequent quantitation of that product using fluorescence, chemiluminescence, or radioactivity [1418]. All of these techniques involve RNA isolation, reverse transcription-PCR (RT-PCR) amplification, and subsequent detection by gel-based or ELISA-type assays. While certain steps of these assays can be automated, the entire procedures are cumbersome and therefore not ideally suited for HTS.

RNA amplification can also be achieved with the use of nucleic acid sequence based amplification (NASBA) [19]. The NASBA technique, unlike assays involving PCR, is isothermal and uses the concurrent enzymatic activities of reverse transcriptase, RNase H, and T7 RNA polymerase, along with two complementary o-nt primers. NASBA-amplified RNA can then be detected by heter-ogenous methods, such as an enzyme-linked gel assay (ELGA) [20], or electrochemiluminescence [21], or by homogenous methods such as fluorescence correlation spectroscopy [22] or molecular beacon probes [23].

A number of available methods for RNA detection do not involve product amplification prior to detection. For example, Wu et al. [24] described an in vitro transcription HTS filtration assay that incorporates multiple radioactive labels in the product transcript. This assay was used to perform a HTS of nonspecific transcriptional elongation by E. coli RNA polymerase (RNAP). In addition, several sandwich hybridization methods have been described that use chemilumines-cence or bioluminescence detection [25,26]. One variation of the sandwich hybridization technique is the sensitive branched DNA (bDNA) signal amplification assay for RNA detection [27]. The bDNA assay utilizes a DNA hybridization probe that is linked to a branched o-nt complex whose ends are labeled with alkaline phosphatase (AP). Thus the readout signal is amplified by the presence of multiple AP labels per hybridization probe. This technique, while automatable, is time-consuming and expensive.

A number of homogenous methods have been described for RNA detection. One such method is the combination of RNase protection and scintillation proximity assay technology [28]. Other homogenous techniques include nucleic acid binders used in the development of assays that allow quantitation of RNA using either fluorescence (e.g., thiazole orange and oxazole yellow) [29,30] or chemilu-minescence detection using acridinium esters [31]. RNA synthesis can also be measured by incorporating fluorescently labeled nucleotides into the nascent transcript [32].

B. Techniques Used for RNA Quantitation in Cell-Based Assays

The most commonly used methods for cell-based HTS of mRNA expression involve reporter gene assays. Typically, the promotor region and other DNA sequence elements thought to be important for appropriate regulation of the gene of interest are linked to a reporter gene. This reporter gene construct is either stably or transiently introduced into a suitable cell line. This type of assay has the drawback that the construct may not contain all of the essential endogenous elements that regulate the gene of interest in its physiological milieu. Stable knock-in cell lines remedy this shortcoming by inserting a reporter gene into the intact endogenous gene. However, the generation of these knock-in cell lines is extremely tedious and often not possible. Furthermore, establishing stable cell lines is a time-consuming process, and appropriate gene regulation is not guaranteed in transformed cell lines.

Commonly used reporter genes used to monitor gene expression are those encoding proteins for which substrates yielding luminescent products are available, and include luciferase, P-galactosidase, chloramphenicol acetyltransferase, and AP [33]. In most cases, the choice of reporter gene is dictated by the nature of the host cell and the level of sensitivity required in the assay. One of the most exciting recent developments in the development of reporter genes has been the use of green fluorescent protein (GFP) from the jellyfish Aequorea victoria [34]. GFP emits green light upon exposure to UV or blue light. Unlike other bioluminescent molecules it does not require other cofactors or substrates, hence simplifying the assay and making it more amenable to HTS. Besides the wild-type GFP, a number of mutants are now commercially available that emit light at different wavelengths, making it possible to perform multiple gene expression assays from the same cells or mixture of cells [35]. Furthermore, because detection of GFP is noninvasive it is well suited to monitor kinetics of gene expression.

Radioactive and nonradioactive in situ hybridization (ISH) is widely used to measure cellular mRNA abundance and localization [36,37]. However, current ISH methods are technically not suitable for high-volume applications, due to the extensive sample manipulation involved. Recently, Harris et al. [38] described a microtiter plate ISH assay utilizing radiolabeled riboprobes that is more sensitive than conventional Northern blots. However, the assay requires a number of steps and is time-consuming (overnight incubation is required for hybridization of the riboprobe to the target mRNA). This protocol is thus not ideally suited for HTS.

Despite these tremendous advances in the field of nucleic acid detection, very few assay formats allow the sensitive detection of RNA in an automated, high-throughput manner. In general the available methods for RNA detection generally lack sensitivity, require extensive manipulation, and are quite expensive. There is clearly a need for methods that allow direct monitoring of mRNA expression in a HTS format without RNA amplification or the introduction of reporter genes.

A novel, sensitive, and simple assay for the detection of specific in vitro transcription reaction products and a facile ISH assay using anti-RNA:DNA hybrid antibodies for the direct detection of mRNA in intact cells is described below. Anti-RNA:DNA antibodies have been widely used for the detection of nucleic acids, particularly in clinical applications [39-41]. Recently, Tropix Inc. has introduced a commercial mRNA HTS assay that utilizes anti-RNA:DNA antibodies to detect specific mRNA expression in cells, which does not need amplification, purification of mRNA, or the development of stable cell lines, which is required with reporter assays. This method involves cell lysis and denaturation, and subsequent hybridization of the target mRNA to complementary DNA-probe (biotiny-ated o-nt). The DNA/RNA complex is then captured in a sterptavidin-coated microplate. An AP conjugated anti-RNA: DNA antibody is added, which binds to the RNA/DNA complex. AP activity is detected using chemiluminescent AP substrate.

Various strategies have been employed to raise monoclonal and polyclonal antibodies that specifically recognize RNA:DNA heteroduplexes independent of the nucleic acid sequence, while not binding to single-stranded or double-stranded RNA or DNA [42-45]. Information from these studies was used to develop the HTS assay for RNA detection described herein.

III. DETECTION OF PURIFIED RNA BY ANTI-RNA:DNA ANTIBODIES

RNA detection assay was optimized and validated by quantifying purified RNA generated from the in vitro transcription of interlukin-8 (IL-8 plasmid containing the human IL-8 cDNA IL-8) and G-less cassette encoding plasmids (G-less plasmid and LTR#5 plasmids contain the HIV LTR promotor with TAR sequences followed by a 450 nucleotide (nt) G-less cassette). The procedure used for mea suring the purified in vitro transcribed RNA is optimized for a number of parameters, including hybridization buffer contents, hybridization time, o-nt concentration, antibody concentration, and wash buffer. Luminescence signal is measured by reading the plates in a luminometer (Victor from Wallac Inc., Gaithersburg, MD).

Data that validate the assay procedure and show that it can detect specific RNA sequences is shown in Figure 1. Panel A shows that the maximum luminescence signal was obtained only when both the target RNA and its corresponding complementary DNA (cDNA) oligonucleotides (o-nts) were added to the reaction mixture. When either the target RNA or o-nts were omitted, the signal was eliminated. Treatment with RNase A, after hybridization of the o-nts to the target RNA (panel A, column 4) resulted in a very small loss in signal relative to no RNase A treatment (column 1). However, treatment with RNase A prior to addition of the o-nts (column 5) completely eliminated the signal as RNase A is known to degrade selectively single-stranded RNA and not RNA:DNA heteroduplexes. These results indicate that RNA: DNA heteroduplex formation is necessary for signal detection. Figure 1, panel B shows that the assay detects specific RNA sequences. The maximum signal was obtained when the target RNA was hybridized to its DNA o-nts (columns 1 and 3); no signal is detected when non-complementary DNA o-nts were used (columns 2 and 4).

The dose-response curve obtained with IL-8 RNA (Fig. 2) shows that the assay is sensitive enough to detect as little as 100 attomoles and is linear up to at least 100 fmoles of RNA (data for the upper end of the range is not shown). The sensitivity and linearity range of the assay make it highly suitable for quanti-tating the RNA product of in vitro transcription reaction. The yield of an in vitro transcription reaction is typically in the low attomole to fmole range. Assay sensitivity is presumably dependent on the amount of RNA:DNA heteroduplex formed, as this creates binding sites for the anti-RNA: DNA antibody. The extent of RNA:DNA heteroduplex formation is dependent on the length and sequence of the target RNA and DNA o-nts, and on the hybridization conditions. RNA sequences typically have extensive secondary structure, which could prevent effective hybridization at temperatures below the melting temperature (Tm). The total number of distinct DNA o-nts used and the regions of the target RNA to which they hybridize were found to be critical for maximizing signal intensity. In order to achieve the maximum signal and yet not have to optimize the sequences of the DNA o-nts used for each target RNA, a number of short DNA o-nts (35-mers) that were contiguous and complementary to the target RNA sequence, and spanned at least three-quarters of the target RNA sequence, are used. Another parameter found to be important for maximizing signal was the hybridization temperature. The hybridization reactions for all of the in vitro transcription experiments are carried out at room temperature, to avoid extra steps of placing

Figure 1 Dependence of specific target RNA detection on cDNA o-nts. These data represent the detection of purified IL-8 or G-less RNA under various assay conditions. (A) Column 1, both the IL-8 RNA (5 fmoles) and IL-8 cDNA o-nts (o-nts) present; column 2, IL-8 RNA (5 fmoles) alone with no cDNA o-nts present; column 3, IL-8 o-nts alone with no IL-8 RNA present; column 4, hybridization of the IL-8 cDNA o-nts to IL-8 RNA (5 fmoles) and subsequent treatment with RNase A; column 5, treatment of the IL-8 RNA (5 fmoles) with RNase A prior to hybridization with the IL-8 o-nts. (B) Column 1, both the IL-8 RNA (5 fmoles) and IL-8 cDNA o-nts present; column 2, IL-8 RNA (5 fmoles) and cDNA G-less o-nts present; column 3, G-less RNA (5 fmoles) and cDNA G-less o-nts present; column 4, G-less RNA and IL-8 cDNA o-nts present.

Figure 1 Dependence of specific target RNA detection on cDNA o-nts. These data represent the detection of purified IL-8 or G-less RNA under various assay conditions. (A) Column 1, both the IL-8 RNA (5 fmoles) and IL-8 cDNA o-nts (o-nts) present; column 2, IL-8 RNA (5 fmoles) alone with no cDNA o-nts present; column 3, IL-8 o-nts alone with no IL-8 RNA present; column 4, hybridization of the IL-8 cDNA o-nts to IL-8 RNA (5 fmoles) and subsequent treatment with RNase A; column 5, treatment of the IL-8 RNA (5 fmoles) with RNase A prior to hybridization with the IL-8 o-nts. (B) Column 1, both the IL-8 RNA (5 fmoles) and IL-8 cDNA o-nts present; column 2, IL-8 RNA (5 fmoles) and cDNA G-less o-nts present; column 3, G-less RNA (5 fmoles) and cDNA G-less o-nts present; column 4, G-less RNA and IL-8 cDNA o-nts present.

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