In Vitro Transcription RNA Synthesis
While there has been significant demand for ultra long RNA oligonucleotides for gene structure and functional studies, the synthesis of long RNA fragments using the conventional RNA synthesis method is highly sequence-dependent. This is due to the potential for secondary structure formation and the inherent difficulty for chemical synthesis with additional 2'-OH reactive group of RNA, as it introduces considerable complexity to the synthesis. The synthetic chemical RNA synthesis method can only synthesize long RNA oligos under 250 bases. Bio‑Synthesis has met the challenge of synthesizing thousands of semi-synthetic long RNA Transcripts >3000 bases by employing expert molecular biologists, gene synthesis technology, and proprietary methods to optimize the RNA manufacturing process in vitro using SP6 or T7 polymerase.
Each in vitro transcribed long RNA oligo is meticulously monitored during synthesis and controlled according to Bio‑Synthesis’s stringent quality assurance and quality control standards. For over 30 years, Bio‑Synthesis has maintained these gold standards for long RNA transcription synthesis that saves the researcher a great deal of time and trouble for applications that require direct synthesis of the entire target fragment
such as mRNA.
These long oligos can be used in variety of molecular biology applications such as:
- RNA control template for Real-Time PCR
- Antisense technology
- Internal controls for house-keeping genes
- External RNA spike-in control assays
- In-situ Hybridization RNA probes
- Expression control via anti-sense RNA
- RNA template for in vitro translation
- RNA structural studies (Protein-RNA binding)
- Ribozyme biochemistry
- Aptamer discovery(SELEX)
RNA Long Oligonucleotide as Synthetic Pre-miRNA-Based shRNA
Bio‑Synthesis provides custom RNA Transcripts by using synthetic DNA templates prepared by us or plasmid DNA supplied by our customer. After RNA synthesis we perform two purification processes. First, phenol-chloroform extraction and ethanol precipitation. This method is use to remove proteins and most of the free nucleotide. Second, Spin column chromatography to remove additional unincorporated nucleotides, proteins and salts. For applications such as labeled RNA probes for RNase protection assay or foot printing experiments which require extremely pure RNA transcript, we recommend gel purification as an optional service.
We provide messenger RNA (mRNA) synthesis and modifications
with flexible synthesis scales for your individual research needs. Our RNA transcription service includes:
RNA Transcription Services
- Sequence design
- Gene synthesis
- DNA purification and quantification
- DNA sequencing verification
- Plasmid linearization
- IVT reaction followed by DNase I treatment
- RNA purification and quantification
- RNA size check (denaturing PAGE or agaros gel)
- Quality assurance certificate
- Formulated in RNaseSAFETM Tube
RNA Transcription Packages
Using SP6 (or T7) polymerases, we can prepare RNA transcripts ranging in size from 50 nts to >5000 nts in length, in quantities from 10 ug to multi-milligram scale.
- QC by PAGE or agarose >80%
- Guaranteed irrelevant signal is < 1% vs. target (equivalent to 7 Ct difference in qPCR assay)
- QC by PAGE or agarose >90%
- Guaranteed irrelevant signal contamination < 0.1% vs. target (equivalent to 10 Ct difference in qPCR assay)
In order to prevent cross-contamination between RNAs we provide:
Other additional analysis include:
- Independent lot-to-lot processing
- QC by PAGE or agarose >90%
- Guaranteed irrelevant signal contamination < 0.01% vs. target (equivalent to 13 Ct difference in qPCR assay)
- Trace DNA Template analysis by PCR
- Template DNA analysis by PCR up to 35 cycles
Purification of RNA Transcripts
All Bio‑Synthesis RNA transcript oligos are purified under RNase free environment.
Purification methods include:
- Column based purification
- Lithium chloride precipitation
- Spin column chromatography
- Phenol extraction followed by alcohol precipitation
- Gel Purification optional
Scales and Yields
Bio-Synthesis provide various synthesis scales with yield ranging from µgrams to multi milligram quantity. Our RNA transcripts quotes are based on starting synthesis scale. The due to the high degree of variability in from template to template. These variations are influenced by sequence, length and base composition. Incorporation of modified bases will also affect final yield. Some modifications, like 5-Methyl-C have little effect upon final yield while 2' Fluoro modifications can have a large effect. The expected yields of RNA transcription in the table below are for 1-2 kilobases. These are not final guaranteed amounts.
All RNA transcript are produce under strict quality management system from acquire reagent from qualify vendor to the production of oligonucleotide to DNA templates. Each step is screened by mass spectrometry, agarose size check and bidirectional DNA sequencing in multiple locations. After transcription, the RNA is carefully checked using denaturing PAGE electrophoresis, Real-Time PCR and other additional custom assay developments as required by our customers. If you are dissatisfied and report an oligo problem within 30 days of order receipt, we will gladly remake your oligo free of charge or provide a credit for the purchase amount. All orders are shipped with a certificate of analysis and other fully traceable documentation.