Description
TeloPrime Full-Length cDNA Amplification Kit V2
The TeloPrime Full-Length cDNA Amplification Kit V2 is an all-in-one protocol for generating full-length cDNA from total RNA.
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 V2 (Cat.No.018.16) containing 16 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

In an mRNA-Seq experiment, mRNAs were tagged at their 5’ end using TeloPrime, Template-Switch (TS) or Oligo Capping (OC). Transcript start sites (TSS) were mapped to the mouse genome. The accumulated TSS read coverage is plotted versus the normalized annotated transcript length to show relative TSS mapping for the top 500 expressed genes.
Workflow
The TeloPrime Full-Length cDNA Amplification Kit V2 is a protocol for generating full-length cDNA from total RNA. It is based on Lexogen´s unique Cap-Dependent Linker Ligation (CDLL) and long reverse transcription (long RT) technology, and is highly selective for full-length RNA molecules that are both capped and polyadenylated.
For viewing the whole workflow on page please click here
Featured Publications
FAQ
Frequently Asked Questions
Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.
Tagging of the 3’ end of the mRNA takes place during the reverse transcription through the use of oligodT priming.
For long-read sequencing applications we recommend using higher input amounts of 1 – 2 µg of total RNA. See also Appendix A (p.20) of the TeloPrime V2 User Guide for more details.




The TeloPrime Full-Length cDNA Amplification Kits (013) contain sufficient reagents to perform a qPCR and endpoint PCR for each sample. Lexogen also offers a TeloPrime PCR Add-on Kit (Cat.No. 018.16) for 16 reactions if additional PCRs are required.
TeloPrime V2 and V1 libraries have been successfully sequenced on PacBio Sequel® instruments. For further details see our TeloPrime Publications page, or contact info@lexogen.com.
When TeloPrime cDNA is intended for Iso-Seq™ library preparation for sequencing on Sequel® instruments (Pacific Biosciences), additional adapters must be added to the ends of the cDNA to facilitate sequencing. For these applications we recommend the following:
- Use 1 – 2 µg of total RNA as input for TeloPrime cDNA generation.
- Use the highest possible quality of RNA
- Purify the amplified TeloPrime cDNA after the endpoint PCR using the Ampure PB® Beads from Pacific Biosciences (for further details see also Appendix D (p.26) of the TeloPrime V2 User Guide).
- TeloPrime V2 Iso-Seq libraries are best loaded for Sequel instruments using the diffusion loading method. Loading amounts around 6 pM have produced high outputs for TeloPrime V2 libraries. However, specific loading amounts may need to be adjusted according to the instrument and chemistry version in use.
- Contact Pacific Biosciences for further advice regarding the Iso-Seq™ protocols and sequencing recommendations.
TeloPrime libraries have been successfully sequenced on MinION devices and are featured in several recent publications.
To prepare TeloPrime cDNA for downstream sequencing on Oxford Nanopore Techologies instruments, adapter sequences must first be added to the ends of the cDNA. In general, Oxford Nanopore Technologies kits for DNA library preparation (omitting any DNA fragmentation steps), or PCR barcoding can be used. Contact Oxford Nanopore Technologies for specific advice on the right kit for your chosen application.
The method of cDNA generation using the proprietary Cap-Dependent Linker Ligation (CDLL) has not changed for the V2 protocol.
Sample barcoding can be included in two ways, either during reverse transcription through the use of custom reverse transcription primers (see Appendix D, p.25 of the TeloPrime V2 User Guide), or by adding sample barcodes after full-length cDNA amplification is complete. Sample multiplexing is supported for long-read sequencing platforms as well:
PacBio IsoSeq
Sample barcodes can be included in alternative oligodT-primers and used in place of the standard Reverse Transcription Primer (RTP) for the first strand cDNA synthesis (steps 1 – 3). We suggest to use the sample barcoded RT primer designs provided by PacBio for IsoSeq libraries (https://www.pacb.com/wp-content/uploads/2015/09/Procedure-Checklist-Multiplex-Isoform-Sequencing-Iso-Seq-Analysis.pdf). These primers can be ordered as desalted oligos. The concentration of these oligos in the reverse transcription reaction may need to be optimized within the range of: 12.5 nM – 1.5 uM.
In addition, a custom RP primer will be needed for the Endpoint PCR, to use together with the standard FP primer. This custom RP primer needs to bind the 5’ end of the barcoded RT primers and should have a melting temperature (Tm) matching, or as close as possible to that of the standard FP primer included in the TeloPrime V2 kit.
Oxford Nanopore
Sample barcodes can be introduced onto TeloPrime cDNA after endpoint PCR using the Oxford Nanopore PCR Barcoding Kit (SQK-PBK004). In order to have compatible adapters for the addition of ONT barcodes the RP and FP primers used for the TeloPrime V2 Endpoint PCR need to be modified to include ONT primer binding sequences.
Figure 5 | Schematic for addition of ONT adapters to TeloPrime cDNA for barcoding and ONT sequencing.
The alternative FP and RP primer sequences required are listed below. These can be ordered as desalted oligos and should be used for the Endpoint PCR at a final concentration of 2 uM.
FP-ONT: 5′– TTTCTGTTGGTGCTGATATTGC TGGATTGATATGTAATACGACTCACTATAG –3’
RP-ONT: 5′– ACTTGCCTGTCGCTCTATCTTC TCTCAGGCGTTTTTTTTTTTTTTTTTT — 3’
*bold sequence is the standard FP and RP sequence and the 5’ sequence is the ONT primer binding sequence.
The Endpoint PCR program should include one cycle at 50 degrees annealing temperature, then using 62 degrees annealing temperature for the remaining cycles.
TeloPrime V2 and V2 IsoSeq data generated by Sequel instruments (Pacific Biosciences) can be best analysed using a combination of the IsoSeq3 pipeline from PacBio (available here: https://github.com/PacificBiosciences/IsoSeq_SA3nUP/wiki/Tutorial:-Installing-and-Running-Iso-Seq-3-using-Conda) and the Transcriptome Annotation by Modular Algorithms (for Iso-Seq data), or “TAMA” software package (https://github.com/GenomeRIK/tama), which is specifically modified to run TeloPrime IsoSeq data. TAMA runs on Python 2.7 and there is no need for any installation.
TAMA is regularly updated so please check the Github repository regularly for the most up to date workflows and add-ons.
To run TAMA with Iso-Seq3 the following pipeline steps can be recommended:
- CCS
- LIMA
- (do not run “isoseq3 refine –require-polya”, this is the step with issues) bamtools convert -format fasta -in unpolished.flnc.bam > flnc.fasta
- Poly-A cleanup (run as: python tama_flnc_polya_cleanup.py {fasta} {outfile}; NOTE: the current version does not handle random non A insertions)
- Minimap2 to the genome
- Sort bam file and convert to sam
- Run TAMA collapse
Size selection can be used to enhance the proportion of longer cDNAs in an Iso-Seq™ library for PacBio long-read sequencing. Size selection of TeloPrime V2 cDNA for Iso-Seq™ is optional. If you wish to perform size selection prior to Iso-Seq™ library prep please follow the guidelines in the Iso-Seq™ Protocol and use the Ampure® PB Beads (from PacBio) for all purification steps post-PCR. For the endpoint PCR for TeloPrime the following modifications are suggested so as to enable sufficient material for size selection and downstream library prep:
Size selection is optional for PacBio Iso-Seq™ library preparation. Size selection may enhance the proportion of longer transcripts that can be sequenced at higher depth during the run, but is not essential. TeloPrime V2 Iso-Seq™ libraries have been successfully sequenced with and without size selection. When performing size selection, please follow the instructions provided in the PacBio Iso-Seq™ protocol (https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Iso-Seq-Template-Preparation-for-Sequel-Systems.pdf):
Size selection may not be necessary in the following cases:
- Plant RNA – May have shorter average transcript length. Experienced customers report size selection is not necessary for plant samples.
- When starting total RNA input quality is < RIN 8. Here the input is already more degraded and therefore less longer full-length cDNAs will be generated.
- If normal transcript abundance should be preserved, as size selection alters the relative abundance of longer vs. shorter cDNAs.
If you do choose to perform size selection (to enrich longer cDNA fraction) please consider the following tips:
- Prepare 2 – 4 endpoint PCRs when amplifying the TeloPrime cDNA – for each PCR use e.g. 2 – 4 µl of cDNA as template and adjust PCR cycle number accordingly. A higher starting yield of cDNA is needed for size selection due to repooling of the larger fraction at equimolar level to the shorter fraction. Each individual PCR should produce >600 ng of cDNA if PCR cycles are correctly optimised using the qPCR assay.
- Pool the PCR products and adjust the total volume to 160 µl with molecular biology grade water.
- Take 100 µl of this pool (10 volumes) and purify with 0.4x or 0.5x Ampure® PB beads according to the PacBio IsoSeq protocol.
- Take 60 µl of this pool (6 volumes) and purify with 1x Ampure® PB beads according to the Pacbio IsoSeq protocol.
- Perform QC on each fraction and re-pool the cDNA in equimolar manner according to PacBio protocol and continue with SMRT bell ligation.
The qPCR assay provides the fastest way to determine the optimal cycle number for endpoint cDNA amplification by PCR. It is important not to overcycle libraries so as to avoid reduced complexity of the final libraries. If qPCR cannot be performed (i.e., no access to a qPCR instrument or SYBR Green I dye cannot be obtained), then endpoint testing can be performed instead. This will require additional PCR reactions and purifications, therefore the TeloPrime PCR Add-on V2 Kit would be required (Cat. No. 018.16), along with additional purification reagents (The Magnetic Bead Purification Module Cat. No. 022.96, or 1x Ampure PB® Beads from PacBio can be used; alternatively pooling the PCR products and purifying using columns included in the TeloPrime V2 kits can be performed however this will use up the provided columns provided). Using 1-2 l aliquots of the purified cDNA obtained at step 29, perform parallel endpoint PCRs for a range of cycle numbers: i.e., 14 – 20 cycles (increasing increments of 2 cycles can be used). For best evaluation in detail, purify the PCR products and run an aliquot on Bioanalyzer (or similar) to check the size distribution profile and yield.
Downloads
TeloPrime Full-Length cDNA Amplification Kit V2
User Guide – Release of TeloPrime V2 (17.12.2018)
Send us your publication & get the RNA T-shirt!
Product Flyer – Release of TeloPrime V2 (20.12.2018)
Material Safety Datasheets
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