Biotinylated RNA

Fragmented, biotinylated RNA prepared in this manner was hybridized to the array and the signal developed using streptavidin-R-phycoerytherin. A confocal laser scanner was used for detection. The researchers estimated that they could detect two transcripts per cell based upon labeling efficiency and an estimated 4% mRNA content in total bacterial RNA. Thus, chip detection of labeled transcripts was found to be more sensitive than detection by Northern blot. Specific genes (e.g., basal levels of cinA) undetectable on Northern blots were quantifiable on the microarray. In addition, it was... 

Since SP6 polymerase cannot use bio-rUTP efficiently, it is not suitable for preparing biotinylated RNA probes. 

Biotinylated RNA (see Section 2.3.2 for preparation) or proteina Streptavidine-agarose beads (BRL)b Blocking buffer 20 mM HEPES-KOH, pH 7.6 300 mM KC1 0.01% Nonidet P-40... 

This procedure describes a method for preparing RNA or protein attached to beads aimed for subsequent affinity purification experiments. To bind a biotinylated RNA-protein complex present in a cellular extract, see Section 5.1.6. 

The biotinylated RNA aptamer has been immobihzed on the gold electrode of 10 MHz piezoelectric quartz crystals and the interaction with Tat protein in solution has been studied following the changes in the oscillation frequency of the crystal. 

The selection started with an RNA library of -2 x 10 sequences of 160 nucleotide length." After tethering the RNA to anthracene via long flexible PEG chains, this library was allowed to react for with an excess of biotinylated maleimide. Biotinylated RNA was recovered using immobilized strepavidin, and selection pressure was gradually increased by reducing reaction time and maleimide concentration. After 10 iterations of selection and amplification, the enriched library showed -6,500-fold rate acceleration, compared to the starting library. Individual members of this enriched library were sequenced and assayed for activity. 

These authors used the same strategy for detecting the CRP. However, in this example a biotinylated RNA aptamer with affinity for CRP was immobilized on the magnetic beads (capture probe) and a biotinylated monoclonal antibody antiCRP conjugated with alkaline phosphatase (AP) (detection probe) was used. The electrochemical detection through DPV allowed a detection limit of 2nM in serum samples (Centi et al., 2009). 

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

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