<i>AccuNext</i> 单细胞 / 微量 cDNA 合成及扩增试剂盒

AccuNext Single Cell/Micro cDNA Synthesis & Amplification Kit

Item No.: AG12501 / AG12502
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Price range: ¥5,000 through ¥15,000

Product Description
This product is suitable for full-length cDNA synthesis and amplification of RNA from eukaryotic cells without cell walls (e.g. mammalian cells) or with Poly(A) tails. This product contains all the components required for cell lysis, reverse transcription (RT) and PCR amplification, either directly from 1 to 1000 cells or from 10 pg to 100 ng of total RNA as a template, and is reverse transcribed using an Oligo (dT) primer with a specific junction; it utilizes the terminal transferase activity of the reverse transcriptase enzyme to introduce the cDNA into the 3′ end of the cDNA as it reaches the 5′ end of the mRNA, and introduces the cDNA into the 3′ end of the cDNA as it reaches the 5′ end of the mRNA, and the cDNA is then synthesized and amplified. When the reverse transcriptase reaches the 5′ end of the mRNA, a few template-independent bases are introduced at the 3′ end of the cDNA, and the cDNA with the specific junction is synthesized by the 5′ Template Switching Oligo (TSO). Subsequent PCR amplification with the junction sequences added to the ends of the first strand of the cDNA ensures that the final cDNA library contains the 5′ end of the mRNA, maintaining the original mRNA transcripts. This ensures that the final cDNA library contains the 5′ end of the mRNA, preserves the original expression information of the mRNA transcript, and avoids the preference for the 3′ end.
NGS sequencing of full-length cDNA libraries can be used to analyze gene expression differences, variable splicing, fusion genes, and other genetic regulatory information. The product is compatible with sample volumes from 1 to 9.5 μl and can obtain cDNA amplification products from 2 to 20 ng.
The reaction system of this product has been carefully optimized, please use the reagents contained in this product for cell lysis, reverse transcription ( RT ) and PCR amplification experiments, it is not recommended to change the dosage and concentration of any of the reaction components or to replace the components in this product with other equivalent products, in order to avoid obtaining bad results. If substitution is required, please verify first.
Product Advantages
① High sensitivity: Highly efficient amplification of single cell template or 10 pg RNA template.
② The carefully optimized reaction system avoids the preference of PCR amplification, and the number of genes (including the number of low-expressed genes) is highly detected.
③ Amplification of full-length cDNA by double-ended primers can obtain full transcriptome information, avoiding 5′ and 3′ end preferences.
Product Composition

Package 2-1 is composed as follows (stored at -80°C):

individual parts making up a compound AG12501 AG12502
Control Total RNA* (1 μg / μl) 5 μl 5 μl
5′ Template Switching Oligo 12 μl 48 μl

*: Control Total RNA is 293T Cell Total RNA.

Package 2-2 is composed as follows (stored at -20°C):

individual parts making up a compound AG12501 AG12502
10X Cell Lysis Buffer 228 μl 920 μl
RNase Inhibitor (40 U/μl) 18 μl 72 μl
3′ Oligo (dT) Primer 24 μl 96 μl
5X First-Strand Synthesis Buffer 48 μl 192 μl
AccuNext Reverse Transcriptase (100 U/μl) 24 μl 96 μl
2X AccuNext PCR Mix I 300 μl 1.2 ml
PCR Primers 12 μl 48 μl
Nuclease free water 1 ml 1 ml
Preservation and transportation
Preservation Temperature:
Package 2-1 -80°C Storage
Package 2-2 -20°C Storage
Transportation temperature:
Package 2-1 Dry Ice Transportation (Avoid repeated freezing and thawing.)
Package 2-2 Dry Ice Transportation or -20°C Ice Pack Transportation
Example 1

Control Total RNA full-length cDNA amplification was performed using this kit with an RNA template addition of 10 pg and a PCR amplification cycle number of 17. The results are as follows: Figure A shows the purified product after cDNA synthesis and amplification, which is evenly distributed. Figure 3. B shows the absence of product in the negative control without template.

Figure 3. Agilent 2100 Bioanalyzer results.

Example 2

293T Cell and 293T Cell Total RNA were reverse transcribed and amplified by PCR using this kit. A DNA library was constructed from the PCR products and sequenced using the Illumina NovaSeq 6000 System:

Table 2. 1 ~ 1000 cells major mRNA-Seq data


Table 3. 10 pg ~ 10 ng of 293T Cell total RNA major mRNA-Seq data

Example 3

Reverse transcription and PCR amplification were performed with the kit using 1~1000 293T Cells as templates, and then a DNA library was constructed from the PCR products, which were sequenced using the Illumina NovaSeq 6000 system and repeated twice. (Figure 4: A is 1000 cells, B is 100 cells, C is 10 cells, D is 1 cell).


Figure 4. Gene expression repeatability analysis

Example 4

1~1000 293T Cells were used as templates for reverse transcription and PCR amplification with the kit, and then the PCR products were used to construct DNA libraries, which were sequenced using the Illumina NovaSeq 6000 system. After sequencing, the distribution of the sequenced fragments was analyzed, and the results showed that there was no preference for 5′- and 3′-end of the fragments amplified with the kit, and the distribution of the fragments was homogeneous (Figure 5). The results showed that the fragments amplified by this kit had no preference for the 5′ and 3′ ends and were uniformly distributed (Figure 5): A is 1000 cells, B is 100 cells, C is 10 cells and D is 1 cell).


Figure 5. Distribution of sequencing data over genes

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