Synthesis of custom designed, long RNA or DNA sequences for in vitro diagnostics tools
- Reference RNA and DNA for development of in vitro diagnostics assays
- Companion diagnostics
- Internal control RNA
- Positive control RNA
RNA available at GMP grade; DNA and RNA available at research grade (ISO13485 certificate)
Synthetic internal control RNA
A negative result in the PCR amplification/detection step can be misleading. False negative results can be caused by failure (i) already in the initial RNA isolation step, (ii) in the reverse transcription, cDNA synthesis step, and finally (iii) in the PCR step.
The use of an internal control DNA can only reveal problems in the last step. The use of customized internal control RNAs allows monitoring of all three critical steps!
- Internal Control RNA with non-target sequence & size
- Monitor all steps in RT-qPCR tests and avoid false negatives
- Internal Control RNA is included before RNA isolation and before cDNA synthesis
Agarose gel electrophoresis of RT-PCR products Internal Control RNA (IC) is spiked into all samples, containing logarithmically increasing amounts of target RNA: 100 to 105 copies.
Negative controls: only IC is amplified; high target amounts outcompete IC amplification (lanes 103 to 105).
Fuzzy primer dimer band at the bottom decreases with increasing specific amplifications.
PCR amplification / Real-time IC and Target detection with two different reporter dyes.
ABI instrument SDS 7700 and TaqMan probes Left panel: IC in JOE channel to detect potential problems in (i) the RNA isolation, or (ii) inhibition in the RT or (iii) in the qPCR step.
No inhibition: All samples show strong signals for IC. Right panel: Target amplicons in FAM channel are positive (red, green, blue, purple, dark blue curves) or negative (dark green base line).
Synthetic Positive Control RNAs
Serial dilution of high quality positive controls is a basic tool for establishing PCR test systems. Furthermore, an important aspect in the development of pathogen detection assays is the evaluation of test specificity: only the intended target sequence(s) must be shown to give positive results, whereas closely related species must be shown to give negative results. The limited availability of known target sequence variants or the samples of closely related species can be challenging.
These requirements for access to complete sequence panels can be challenging for new test developers who do not have the resources of established units. The availability of customized reference sequences offers unlimited possibilities for all test developers.
This also applies to specific transcript detection assays with rare splice variants or sequence mutations as targets.
The use of Synthetic RNAs makes the development of mutant screening tests straightforward and any desired sequence variant is available in homogeneous form, independent from the limited availability of (uni-variant) biological samples.
The implementation of Synthetic Positive Control RNAs in all routine test runs provides routine performance control of the tests and guarantee maintenance of high quality standards.
Dilution series of Target Nucleic Acid. RotorGene 3000 instrument and TaqMan probes
Left panel: Amplification curves, all reactions were run in triplicates.
Right panel: Graphic quantification plot
Positive Control RNAs for evaluation of real-time RT-qPCR assays or DNAs for qPCR assays
The Full Service includes:
Synthesis of synthetic DNA, plasmid cloning, purification, full DNA sequencing documentation, PCR amplification of insert, purification and characterisation with subsequent RNA synthesis by in vitro transcription, purification and characterisation.
The customer provides text files with the desired sequence data.
- Chemical synthesis of dsDNA fragments
- Cloning of the DNA fragments in a plasmid
- Quality control by sequencing of plasmid inserts
- Synthesis of PCR primers, based on the plasmid sequence, for PCR amplification of the cloned insert sequence
- PCR amplification of the cloned insert with the primers described in step 4
- Purification of the PCR products with spin columns
- Quality control of PCR products by capillary electrophoresis (Agilent Bioanalyzer)
STOP here for PCR product DNA
- PCR templates for production of ssRNA by in vitro transcription with T7 RNA polymerase
- DNase treatment for removal of the DNA template
- Purification of ssRNA with spin columns
- Quality control of the in vitro transcripts by capillary electrophoresis (Agilent Bioanalyzer)
- Quantity: 5 μg of ssRNA