RNA probes

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

Recent innovations for detecting malaria include DNA or RNA probes by polymerase chain reaction (PCR). These, however, are not widely available for clinical use. A rapid dipstick test (ParaSight F, Becton-Dickinson, Cockeyville, MD) reportedly has a sensitivity of 88% and a specificity of 97%, which is comparable to microscopy. However, ParaSight F can give false-positive results with rheumatoid factor thus microscopy remains the optimal test. 

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

In situ hybridization Use of a DNA or RNA probe to detect the presence of the complementary DNA sequence in cloned bacterial or eukaryotic cells. 

The RPA is a sensitive method for quantifying specific RNAs from a mixture of RNAs. This is achieved using a small-volume hybridization of an RNA probe to the RNA under study. Unhybridized probe and sample is then digested with RNAses and the protected probe fragment is visualized after denaturing gel electrophoresis. Commonly, the probe is radiolabeled for maximum sensitivity. Following is a method for RPA detection of R-luc-4 sites and F-luc mRNA. 

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cyanine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to synthesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol cyanines containing few or no sulfonates are used in these applications to label oligos for applications such as array detection, hybridization assays, and RT-PCR. 

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. 

The following protocol describes the modification of DNA or RNA probes at their 5 -phosphate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide. A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2 (this chapter). 

Childs, C.V., Lloyd, J.M., Unabia, C., Gharib, S.D., Wierman, M.E., and Chin, W.W. (1987) Detection of luteinizing hormone b messenger ribonucleic acid (RNA) in individual gonadotropes after castration Use of a new in situ hybridization method with a photobiotinylated complementary RNA probe. Mol. Endocrinol. 1, 926-932. 

Using 0.2 kb of the 3 UTR region of hydAl, a RNA probe was generated and hybridised with total RNA which had been derived from 50 ml culture samples after 0, 1, 2 and 4 hours of anaerobic induction. The Northern blot results demonstrate that hydAl is highly transcribed under conditions of anaerobic adaptation (Fig. IB). 

A. Biotin Incorporate in base thymine in DNA probe sequence or uracil in RNA probe sequence... 

An amplification system that actually amplifies exponentially RNA probe sequences bound to the target sequence, in contrast to PCR and TAS systems, which amplify target sequences, is the Q-beta replicase system (B4). Although this system can achieve a million- to billionfold amplification in 15 minutes at 37°C, background signal due to nonhybridized probes is reported to be very high. 

Most protocols that use RNA probes recommend stringent procedures to ensure the absence, as far as possible, of contaminating RNase during the probe preparation and hybridization procedures. RNase is... 

Meltzer JC, Sanders V, Grimm PC, Stern E, Rivier C, et al. 1998. Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization. Brain Res Prot 2 339-351. 

Resuspend the pellet in 6 pi of DEPC-treated ddH O. Use a 1-pl aliquot to determine the concentration with a spectrophotometer and check the quality of the RNA probe on an agarose gel see Note 9). 

Prepare Hybridization Mix containing 500 ng/ml of DIG-labeled RNA probe in a plastic screw-cap tube, and transfer the embryos. 

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