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.
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.
Capped Poly(A) RNA
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.
Capped Poly(A) RNA
with the 5’ tag get extended in the second-strand synthesis.
Capped Poly(A) RNA
full-length cDNA is converted into full-length ds cDNA.
Capped Poly(A) RNA
and 3’ tag specific PCR primers to provide enough material for various
For viewing the whole workflow on page please click here
Frequently Asked Questions
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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.
Figure 1 | TeloPrime cDNA prepared with V2 PCR Protocol for vertebrate and plant RNA. TeloPrime cDNA was generated from 1 µg of total RNA using a custom reverse transcription primer. The cDNA was amplified using a custom PCR reverse primer and the standard Forward PCR primer (FP), with 17 cycles (vertebrate, red trace) or 21 cycles (plant 1, blue trace, and plant 2, green trace) for endpoint PCR. PCR products were purified using 1x AMPure® PB beads (Pacific Biosciences) and normalized to a concentration of 300 pg/ul for analysis on a High Sensitivity DNA Chip (Bioanalyzer, Agilent Technologies). Customer-provided data reproduced with permission.
Figure 2 | TeloPrime cDNA prepared with V2 PCR protocol from sheep total RNA (RIN 8.4). TeloPrime cDNA was generated from 1 µg of total RNA, with standard TeloPrime RT primer and amplified with the standard TeloPrime V2 PCR primers. For endpoint PCR, 1 µl of cDNA was amplified with 26 cycles. The PCR product was purified using TeloPrime purification solutions and columns, and analyzed on a DNA 12000 Chip (Bioanalyzer, Agilent Technologies). Customer-provided data reproduced with permission.
Figure 3 | TeloPrime cDNA generated with different PCR cycles. Endpoint PCR was performed using 2 µl of pooled TeloPrime cDNA with Optimal Endpoint PCR (OEP) cycle number (as calculated from qPCR, i.e., 80 % of maximum fluorescence), or using OEP ± 3 cycles. The amplified cDNA was analyzed on a Bioanalyzer DNA 12000 Chip (Agilent Technologies). Undercycling (green, OEP-3) produces low yields, while overcycling by 3 cycles (red, OEP +3) results in enrichment of shorter versus longer products and also reduces the yield. The optimal cycle number (blue, OEP) gives highest yield for longer cDNAs.
Figure 4 | TeloPrime cDNA Quality Control. TeloPrime cDNA was prepared from 500 ng of Universal Human Reference RNA (UHRR). Amplification was performed using 1 µl of cDNA with 24 cycles of PCR. Final cDNA was run at 1:10 dilution on a Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies). The optimal endpoint PCR (OEP) cycle number was determined by qPCR as described in Appendix B, p.22.
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 firstname.lastname@example.org.
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:
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.
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 Full-Length cDNA Amplification Kit V2
Material Safety Datasheets
TeloPrime Free Trial Kit
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