RNA transcript labeling

Labeled Extract Preparation and Microarray Hybridization BioArray Highyield RNA Transcript Labeling System (Enzo)... 

BioArray high-yield RNA transcript labeling kit (Enzo Diagnostics 42655-10). 

Labeled Extract Synthesis BioArray High-Yield RNA Transcript Labeling System (for 2pg or Total RNA and Above)... 

The BioArray Highyield RNA transcript labeling system can be subdivided into discrete steps (see Fig. 1)... 

Fig. 1. (A) Agarose gel electrophoresis of total RNA (500ng/lane) extracted from five different regions (1-5) of the neural tube of E9.5 CDl mouse embryos using the Trizol protocol detailed in section 3.2. The arrows point to the characteristic 28s and 18s rRNA bands. For intact total RNA, the 28s band should be approximately double the intensity of the 18s band (see Notes 1-11). (B) Agarose gel electrophoresis of the biotin labeled extracts generated by the Enzo BioArray Highyield RNA Transcript Labelling System from 2pg of starting...

The protocol described here is an adaptation from that supplied with the BioArray high-yield RNA transcript labeling kit (see Note 27). Thaw the high-yield lOx reaction buffer and DTT at 37°C and keep at room temperature prior to use. Thaw all other reagents at room temperature and place the enzymes on ice. It is essential that samples and reactions constituting the same microarray study be derived from the same master mix. 

Bio Array High-Yield RNA Transcript Labeling System RNA Labeling In-Vitro Transcription Reaction Clean Up... 

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

Scheme 3.3 Flow-chart for making labeled RNA constructs by enzymatic (T4 DNA bgase) hgation. Denaturing PAGE purifications after each step of RNA transcript preparation are performed, but now shown for clarity.

A high-throughput assay for bacterial RNA polymerase has been successfully developed and validated using a 96-well, automated format [70], The reaction mixture contained a DNA template, nucleotide substrates (NTPs), supplemented with a-33P-labeled CTP in Tris-acetate buffer (pH 6.8). The polymerase reaction was carried out at 34°C for 40 min (providing linear kinetics). The effect of dimethylsulfoxide (DMSO), the usual solvent for test compounds used in a screen, was taken into consideration. The radiolabeled RNA transcripts were allowed to bind diethyl aminoethyl (DEAE) beads, which were then separated via filtration, and radioactivity associated with the wells was quantitated to measure the RNA polymerase activity. The standard deviation of the measured activity was typically < 15% of the average. Use of this assay to screen for RNA polymerase inhibitors from chemical libraries and natural products led to the identification of DNA intercalators (known to inhibit RNA polymerase activity), rifampicin (a known inhibitors of RNA polymerase), and several derivatives of rifampicin from Actinomycetes extracts. Therefore this assay can be reliably utilized to detect novel inhibitors of bacterial RNA polymerase. 

In this experiment, you used a-[32P]ATP as the radiolabeled nucleotide. Would your results have been any different if you had used y-[32P]GTP as the radiolabeled nucleotide Explain your answer in terms of the mechanism of the reaction catalyzed by RNA polymerase. Could you have used another -y-labeled nucleotide and still obtained a radiolabeled RNA transcript Explain your answer. 

For the detection of mRNA (extracted with proteinase K/SDS or SDS), the signal/noise ratio improves 50-100 times when one of the probes is cRNA (particularly label probe) and another 3-5 times when both probes are RNA (transcripts from the proper inserts in vectors) (Tenhunen et al., 1990). They have also determined optimum concentrations (with respect to signal/noise ratio) of DeSO4 and formamide as 2% and 20%, respectively, for cRNA probes. 

Digoxigeiiin Labeling of RNA Transcripts from Multi- and Single-Locus DNA Minisatellite Probes... 

In order to meet the above criteria, RNA transcripts are applied instead of DNA probes and radioactive deoxyribonucleotides are replaced by digoxigenin-labeled ribonucleotides. Transcription by T7 RNA polymerase of a linearized recombinant vector containing the DNA probe and a T7 promoter, in the presence of digoxigenin-... 

---