AccelerRT® 5G Full Length cDNA Synthesis & Amplification Kit

Product Description

The AccelerRT® 5G Full Length cDNA Synthesis & Amplification Kit can synthesize cDNA ranging from 1-1000 cells or 10 pg – 100 ng of total RNA and an Oligo(dT)VN Primer as a reverse primer. Upon reaching the 5′ end of the RNA template, a specific adapter sequence is annealed and extended to the 3′ end of the cDNA by the terminal deoxynucleotidyl transferase (TdT) activity of the 5G Template Switching Reverse Transcriptase. The full-length cDNA is further amplified by PCR with the adapter sequence, which effectively avoids the 3′ bias in the process of cDNA synthesis. The full-length cDNA amplification products can be used to analyze gene expression differences, variable splicing, fusion genes and other genetic regulatory information.

Advantages

• High sensitivity: Low abundance targets can simply be detected from a small number of cells or total RNA
• High quality cDNA: Double-ended primers amplify full-length cDNA, effectively avoiding 5′ end and 3′ end bias
• Time saving: Shorter cell lysis and reverse transcription time
• Wide compatibility: Pre-amplification compatible with downstream analysis of NGS or Real-time PCR

Applications

• First-strand cDNA synthesis for full length cDNA products
• Construction of single cell sequencing libraries
• Discovery and detection of fusion genes
• Single B cell (VDJ) sequence amplification

Template Switching Function

Figure 1. Introduction to the principle of the AccelerRT® 5G Full Length cDNA Synthesis & Amplification Kit. The Oligo(dT) VN Primer is used to synthesize the first-strand cDNA. Upon reaching the 5′ end of the RNA template, a few non-template nucleotides are added to the 3′ end of cDNA (Template-switching) using the terminal deoxynucleotidyl transferase (TdT) activity of reverse transcriptase. The second-strand of the cDNA is synthesized using a template-switching oligo. Finally, the full-length cDNA product is obtained by PCR amplification using 5’-Template-switching oligo (TSO)-specific primer and 3’universal adapter primers.

Workflow Full-length cDNA Amplification

Figure 2. Workflow of evaluating full length cDNA synthesis. Full length cDNA  of cells or RNA were reversed using AccelerRT® 5G Full Length cDNA Synthesis & Amplification Kit. Part of the cDNA product is pre-amplified by PCR, while the other part is not. Then the cDNA product and PCR product were quantitatively analyzed by qPCR using specific primers of different genes. The difference values of CT values indicated that the full-length cDNA was amplified successfully.

Performance Data

Figure 3. Using AccelerRT® 5G Full Length cDNA Synthesis & Amplification Kit for full length cDNA reverse transcription of 10ng Hela RNA. Then the cDNA and PCR products of 22 genes were analyzed by qPCR.

Figure 4.  Various inhibitors were added to total HeLa RNA, then was used in a reverse transcription reaction with AccelerRT® 5G Full Length cDNA Synthesis & Amplification Kit, the synthesized cDNA was used as a template in subsequent qPCR using BlazeTaq™ SYBR® Green qPCR mix 2.0(Cat.# QP031).