Product Description

The TeloPrime Full-Length cDNA Amplification Kit is an all-in-one protocol for generating full-length cDNA from total RNA.

Performance

Full-Length cDNA Synthesis

Based on Lexogen´s unique Cap-Dependent Linker Ligation (CDLL) and long reverse transcription (long RT) technology, it is highly selective for full-length RNA molecules that are both capped and polyadenylated.

For Multiple Downstream Applications

The full-length cDNA products can be used for various downstream applications such as NGS, RACE, cloning, microarray probes, and normalization. It enables the detection and correct quantification of splice variants and their true transcription start- and end-sites, in both short and long mRNA molecules. For further full-length or gene-specific PCRs, Lexogen offers a TeloPrime PCR Add-on Kit (Cat.No.018.30) containing 30 rxn. This kit contains the PCR Forward and Reverse Primer separately,hence they can be alternatively substituted with the gene-specific primer of interest

Exceptional Elimination of cDNA from Degraded RNA

Based on the CDLL technology the ligation of the 5’ linker to the 3’ end of the cDNA takes place in a highly cap-dependent manner, providing an exceptional cap-specificity and eliminating cDNA products from degraded mRNA.

Workflow

Step 1: First Strand cDNA Synthesis (Oligo dT Priming)

Firstly, full-length cDNA synthesis is initiated by oligodT primed long reverse transcription. This helps to preserve the complete RNA sequence information in the cDNA before a cap selection is carried out. In addition a more stable RNA/cDNA hybrid is created that is maintained throughout post RT purification. This double-stranded (ds) hybrid is also important for the specificity of the subsequent Cap-Dependent Linker Ligation reaction.

Step 2: Double-Strand Specific Ligation

The double-stranded adapter with a 5’ C overhang allows for an atypical base-pairing with the inverted G of the cap structure. By using a ds-specific ligase, the ligation only takes place if the cap is present and if the RT has really reached the 5’ end of the mRNA. No ligation takes place if no cap is present e.g., in degraded RNA (low RIN) or if the RT has terminated prematurely because of secondary structures.

Step 3: Second Strand Synthesis

Based on this Cap-Dependent Linker Ligation only products with the 5’ tag get extended in the second-strand synthesis.

Step 4: Second Strand Synthesis

All remaining background is eliminated and the 5’ tagged full-length cDNA is converted into full-length ds cDNA.

Step 5: Amplification of Full-length cDNA

Full-length cDNA is then globally amplified in a PCR reaction using 5’ and 3’ tag specific PCR primers to provide enough material for various downstream applications.

FAQs

Ordering Information

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User Guide – update 01.04.2016 (consistency changes)
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Material Safety Datasheets

MSDS information for TeloPrime Full-Length cDNA Amplification Kit