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Product Information
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.
FAQ
Answer: Experiments for downstream analysis by NGS, preamplified cDNA should be purified to remove residual primers, nucleotides, and enzymes that may affect library preparation. Similarly, pre-amplified PCR products analysis by qPCR were also needed to be purified, then performed the qPCR experiments. However, qPCR analysis of the cDNA product that after RT reaction would not need to be purified, but the cDNA sample should not exceed 1/10 in the reaction system.
Answer: The kit is designed for preamplification of full-length cDNA. It is based on oligo (dT) and in order to obtain full-length cDNA, it is recommended to use high-quality RNA with intact poly(A) sequences at the 3′ ends.
Answer: No need. Because only poly(A) RNA is amplified.
Answer: The following table is the number of PCR amplification cycles based on Hela cell and Hela total RNA, for reference only. The expression of different cells or RNA is different, so the optimal number of cycles needs to be further explored. The high number of amplification cycles will result in the bias of PCR amplification, resulting in the decrease of DNA library volume. The low number of amplification cycles will lead to insufficient DNA amount, which will affect the library analysis.
Answer:
1) Cell sample:
Prepare fresh mammalian cells suspended in a PBS solution and the cells can be lyzed directly using the lysate in this product. The product is not suitable for cells that have been fixed by formaldehyde, acetone, etc. Since this kit uses Oligo dT as primer for reverse transcription, it is not suitable for samples without Poly A tail.
2) RNA samples:
For best results, use high quality Poly(A) RNA and total RNA samples with high integrity and purity. Make sure the RNA does not contain contaminants such as residual proteins, organic solvents, and salts that can degrade the RNA or reduce enzyme activity and sensitivity. Before the experiment, the Agilent RNA 6000 Pico Kit could be used to evaluate RNA integrity, and RNA with RIN≥8 were recommended.
