In-vitro transcription reaction

To obtain milligram quantities of mRNA the previous in vitro transcription reaction is scaled up to a volume of 6 or 12 ml, depending upon the need. 

For the in vitro selection process, the RNA pool containing 10 different sequences and structural motifs is generated by an in vitro transcription reaction. Folding of the RNA molecules is induced by heat denaturation and renaturation at room temperature (26). 

To perform in vitro transcription reaction Save 20 pi of the total volume of 100 pi (for P-UTP transcription for binding studies with uAChRs in Torpedo electric organ membranes, and in case you have to amplify the DNA again) and use 80 pi for transcription. 

Radiolabeled RNA can be generated either by incorporation of a (a32p) nucleoside triphosphate during an in vitro transcription reaction or by the transfer of (y P)-ATP to the 5 terminus of a dephosphorylated RNA molecule (41). The authors prefer the first mentioned method, as it needs only a single enzymatic reaction. 

Ethanol-precipitate the DNAs and resuspend at a concentration of 200 ng/pl for in vitro transcription reactions. 

The purified DNAs are used as templates for in vitro transcription reactions. Forward primer (P-40, same sequence as primer used during SELEX process) and reverse primer (p-22 pGEM). 

Perform the in vitro transcription reaction using the T7 RiboMAX Express Large Scale RNA Production System (tee Note 13) at 37°C for 30 min (tee Note 14). Use 5 pg of DNA template for 100 pL of transcription reaction. Typically, about 500 pg of purified mRNA is obtained from 100 pL of transcription reaction. This yield corresponds to about 1.5 mL of translation reaction. 

Procedures to set up the NICK Columns are as follows. First, remove the column cap and pour off the excess liquid. Rinse the column with 3 mL ofsterilized distilled water. Remove the bottom cap and place it in a column stand. Equilibrate the gel with 3 mL of sterilized distilled water and flush completely. These procedures should be carried out during the in vitro transcription reaction. 

Discard the supernatant and then rinse the pellet with 70% ethanol. Do not dry the pellet completely, so it will dissolve mRNA in water easily. Dissolve the pellet in 100 pL of sterilized distilled water. If the reaction scale of the in vitro transcription is less than 100 pL, dissolve the pellet with sterilized distilled water in an equal volume of the in vitro transcription reaction. 

At least 125 pg/mL of the linearized DNA template is required for in vitro transcription reactions. 

Avoid using PCR polymerases that generate 3 overhangs. This kind of overhang can result in the decreased efficiency of the in vitro transcription reaction. 

Figure 2.2 Schematic diagram of in vitro transcription reaction. The telomerase RNA gene is cloned behind a T7 RNA promoter. PCR is used to generate linear DNA and the PCR amplicons are in vitro transcribed with T7 RNA polymerase. The RNA is purified away from free nucleotide on a desalting column.

In this experiment, you will carry out an in vitro transcription reaction from a plasmid (pSP72, Fig. 22-4) containing a T7 RNA polymerase specific promoter. A 32P-labeled nucleoside triphosphate (labeled at the a position) will be included in the reaction to produce a radioactive RNA molecule. The size and sequence of the various transcripts that you will produce can be predicted by the restriction enzymes that the plasmid will be digested with prior to the beginning of the transcription reaction. Since the plasmid template for the transcription reaction will be digested, you will be preparing runoff transcripts from the T7 promoter. As a result,... 

Determine the exact sequence of the transcripts that you would expect to be produced if the same templates used in this experiment (pSP72 digested with Bam III, Pro RI, and Hindlll) were subjected to an in vitro transcription reaction using the SP6 RNA polymerase rather than the T7 RNA polymerase. 

Prepare plasmid DNAs for in vitro transcription reactions by digestion with the appropriate restriction enzyme using buffers as provided by the vendor. 

Because of the lack of a reliable quantitative assay to control for the presence of the Cap structure, aside from a functional assay of the mRNA expression (transfection of the mRNA in cells and detection of the translated protein), the enzymatic capping of mRNA is rarely used to produce mRNA for research or therapy. The utilization of the synthetic Cap (nonmodihed analog or ARCA) in the in vitro transcription reaction is the standard method to produce capped mRNA. 

The result is high-quality arrays that can be used with a fluorescence detection system in a manner similar to the glass slide microarrays, although only single color detection is used. cDNA is synthesized from a test RNA sample by reverse transcription followed by second strand synthesis. This is then used as a template for an in vitro transcription reaction to produce biotin-labeled cRNA. As well as introducing the label for detection, this step permits amplification of the RNA and thus enables less starting material to be used. The cRNA is hybridized to the GeneChip and the biotin detected with an avidin-... 

In-vitro transcription reaction (production of biotin labeled cRNA). 

BioArray High-Yield RNA Transcript Labeling System RNA Labeling In-Vitro Transcription Reaction... 

Add 60 pL of RNase-free water to the in-vitro transcription reaction and mix by vortexing for 3 sec. 

A standard in vitro transcription reaction (5) incorporating digoxygenin-11-UTP is performed. SP6, T7, or T3 RNA polymerase may be used. Temperature sensitive reagents should be kept on ice, but the reaction should be mixed at room temperature to avoid precipitation of the DNA template. Reaction ... 

Analogously, the d5SICS-dNaM unnatural base pair developed by Romesberg et al. [107, 142] tolerates amino-modifications at both components of this unnatural base pair with sufficient efficiency in in vitro transcription reactions to allow postsynthetic RNA labeling using NHS-ester chemistry [111, 143]. As a first example of post-transcriptional functionalization of a long RNA, a site-specifically labeled 77 nucleotide amber suppressor tyrosyl tRNA from Methanocaldococcus jannaschii was synthesized via in vitro transcription and post-transcriptionally reacted with NHS-biotin [143]. 

Fig. 3 Unnatural base pairs for enzymatic site-specific labeling of oligonucleotides using click chemistry, a DNA labeling using the unnatural d5SlCS-dNaM base pair [120]. PCR amplification was carried out with an alkyne-modified dfiSICS nucleoside that can be reacted with azide containing compounds, b RNA labeling via T7 in vitro transcription. The functionalized unnatural triphosphate is site-specifically incorporated into an RNA transcript in an in vitro transcription reaction and post-transcriptionally labeled via click chemistry. Triphosphates of the depicted unnatural bases for copper-catalyzed (Eth-C4-Pa [135]) and copper-free click reactions (N3-Pa [152] and Nor-UB [144]) have been developed...

Likewise, Hirao et al. applied copper free chck chemistry for RNA functionalization via the 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbalde-hyde (Pa) unnatural base pair [152], For this, an azide-modified Pa nucleobase (N3-Pa) was incorporated as triphosphate in a T7 in vitro transcription reaction using Ds as corresponding nucleobase in the DNA template (Fig. 3b, 2). Post-transcriptional modification was achieved using fluorescent dibenzocyclooctyne (DIBO) derivatives in a strain-promoted azide-alkyne cycloaddition reaction. Transcription and efficient site-specific labeling of a 260mer RNA was demonstrated. 

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