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Long RNA Synthesis by In Vitro Transcription

Whether you need custom long RNA transcription, or chemical RNA synthesis as molecular control or CRISPR genome editing, Bio-Synthesis provides solutions which are tailored to meet your needs. We provide long custom RNA synthesis by chemically synthesized RNA oligonucleotides or In vitro transcription RNA Synthesis using template options such as plasmids, PCR products, oligonucleotides or cDNA. Our Long RNA Transcription Synthesis services include design as well as gene synthesis using plasmid containing a bacteriophage promoter (such as T7, T3 or SP6). Synthesizing long RNA by in vitro transcription is an alternative to chemically synthesized RNA which is expensive, offers reduced yield and quality, and is limited in length (up to 200 nt.). By contrast, long RNA transcription synthesis obtained from bacteriophage, produces high quality RNA that is thousands of nucleotides long. RNA modifications such as fluorescent labels, end capped modifications or modified RNA bases are also available. Our track record and expertise in providing high quality long RNA transcription products is unmatched by other suppliers.

We are committed to ISO Total Quality Management (TQM) guidelines. All Custom Long RNA Transcription Synthesis is delivered purified using various purification options. Our quality assurance includes bidirectional DNA sequencing for each inserted clone, purity check of dsDNA and RNA by electrophoresis, and/or real-time PCR, qPCR analysis. RNA transcription products are delivered normalized and packaged lyophilized in an RNaseSAFE TM tube to deactivate trace RNase in the liquid phase and also provide an added level of RNA stability during sample handling, with or without ice. Our long RNA transcripts are ideal for functional gene analysis and the development of medical therapeutics and diagnostic needs.

Contact us for Long RNA Transcription Synthesis Services.

Highlights of RNA Transcription Services

  • Unmodified RNA transcripts: Suitable for biochemical assays, structure studies or used as RNA probes.
  • Modified bases: 5-Me-CTP, pseudo-UTP and other modified NTPs
  • Terminal Capping: m7G(5')ppp(5')G (mCAP, Anti Reverse Cap Analog (ARCA), poly A or poly(dT), fluorescent dye incorporation.
  • Other modifications: Poly A or poly(dT), fluorescent dye incorporation.
  • Process optimization: For scales up to multi-gram quantity
  • High Quality: Stringent quality control with optional custom assay development and validation
  • Flexible Solutions: Transcripts from plasmids, PCR products, oligonucleotides and cDNA. Delivered normalized and lyophilized
Long RNA Transcription Synthesis
  • Left: Ambion's Millennium Marker
  • Right: mecA RNA product

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.

Applications

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
  • Subcloning
  • 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.

Standard package:
  • >85% purity verified by PAGE or agarose
  • Guaranteed irrelevant signal is < 1% vs. target
Silver package:
  • >90% purity verified by PAGE or agarose
  • Guaranteed irrelevant signal is < 0.1% vs. target
Gold package:
  • >95% purity verified by PAGE or agarose
  • Guaranteed irrelevant signal is < 0.01% vs. target

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.

Transcription Scale RNA Expected Yield
(uncapped, no poly(A) tail)
mRNA Expected Yield
(capped, poly(A) tailing)
Plasmid DNA Requirements
0.1 ml 0.02 - 0.3 mg 0.01 - 0.1 mg ~15 ug
0.5 mL 0.5 - 1.5 mg 0.25 - 0.75 mg ~50 ug
1.0 ml 1 - 3 mg 0.5 - 1.5 mg ~100 ug
2.0 mL 2 - 6 mg 1 - 3 mg ~200 ug
4.0 mL 4 - 12 mg 2 - 6 mg ~400 ug
6.0 mL 6 - 18 mg 3 - 9 mg ~600 ug
10 mL 10 - 30 mg 5 - 15 mg ~1000 ug

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.

Commonly Asked Questions:

Reference/Citing:

  1. K Terasawa et al. " Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers." Journal of Nucleic Acids, Volume 2011 (2011), article ID131479
  2. B. Wang et al. "Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example." Virology Journal 2011, 8:405
  3. F. Yang et al. "Long non-coding RNA high expressed in hepatocellular carcinoma (lncRNA-HEIH) facilitates tumor growth through enhancer of zeste homolog 2." Hepatology, 2011.
  4. C. Braconi et al. " MicroRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer." Oncogene. 2011, 1-7

Ordering & Contact Information

  • Please contact us for additional information or send an email to info@biosyn.com.
  • You may also request an online quote.
  • To contact us by phone, please call 1-972-420-8505 or Fax at 1-972-420-0442
  • Orders may be placed using a purchase order (PO) or by credit card through our secure online ordering system.
  • When using a credit card ( creditcard ) it will be billed under "Bio-Synthesis, Inc."