Our in vitro transcription vectors are simple and efficient systems for RNA synthesis, applicable for a variety of research purposes. The in situ hybridization version of the in vitro transcription vector system is ideally suited for producing RNA probes (i.e. riboprobes), for use in in situ hybridization assays.
This system incorporates both a T7 and an SP6 promoter with your sequence of interest cloned between these two promoters. Under appropriate reaction conditions and in the presence of nucleotide triphosphates, either T7 bacteriophage RNA polymerase (RNAP) or SP6 RNAP can facilitate highly efficient production of riboprobes corresponding to your sequence of interest. These two promoters have opposing orientations, such that one promoter will produce a “sense” transcript while the other will produce an “antisense” transcript. Depending on the orientation of the insert and the experimental plan, the user may choose to perform in vitro transcription using either T7 RNAP, SP6 RNAP, or both. The flanking T7 and SP6 promoters can also be used as priming sites for PCR amplification or sequencing of inserted sequences. During in vitro transcription, hapten-labeled, fluorophore-labeled, or radiolabeled nucleotides may be incorporated to allow detection of probe localization for in situ hybridization assays. We recommend that you follow established in vitro transcription and in situ hybridization protocols available in published literature.
T7 and SP6 RNAPs both have certain base requirements for efficient transcription initiation which have been already incorporated into the vector design. When T7 RNAP is used, the first two nucleotides of the RNA transcript will be GG, corresponding to the 3’-end of the T7 promoter sequence, followed by your transcript sequence of interest. When SP6 RNAP is used, the first three nucleotides of the RNA transcript will be GAA, corresponding to the 3’-end of the SP6 promoter sequence, followed by your transcript sequence of interest.
Our in vitro transcription vector for in situ hybridization is engineered for run-off transcription. This means that T7 and SP6 RNAPs proceed to the end of the DNA template, and do not terminate at any specific sites within the plasmid. For this reason, the circular plasmid template should be linearized by restriction digestion prior to in vitro transcription. This vector system includes unique restriction sites for BsiWI, AgeI, and AscI next to the T7 promoter, and AvrII, XhoI, NotI, and SapI sites next to the SP6 promoter. Any of these enzymes will cut the plasmid at unique sites immediately downstream of your sequence of interest. Care should be taken that the sequence to be transcribed does not contain any restriction sites for the linearization enzyme used, as this would result in truncated mRNA transcripts. Contaminants from the digestion reaction may inhibit the subsequent in vitro transcription reaction, so purification via column or phenol:chloroform extraction following digestion is recommended. However, it is generally not necessary to purify the promoter and insert fragment away from other fragments, because only the fragments containing the promoter sequences will serve as templates for transcription.
For further information about this vector system, please refer to the papers below.