Custom cDNA Synthesis

BOC Sciences offers cDNA synthesis that uses RNA as a template to obtain cDNA by reverse transcription. The cDNA can then be used as a template for various downstream applications in RNA research.

What is cDNA?

cDNA (complementary DNA) is synthetic DNA whose base sequence is complementary to that of DNA. cDNA is synthesized by the action of RNA-dependent DNA polymerase (reverse transcriptase) in the presence of appropriate primers using RNA as a template, and after synthesizing a single-stranded cDNA and then treating it with a base to remove its corresponding RNA, it is then synthesized by the action of DNA-dependent DNA polymerase using the single-stranded cDNA as a template. After single-stranded cDNA is synthesized and its corresponding RNA is removed by alkali treatment, single-stranded cDNA is used as a template to synthesize double-stranded cDNA by the action of DNA-dependent or RNA-dependent DNA polymerase.

Fig.1 Procedure for cDNA synthesis. (Meis et al., 2009)

BOC Sciences' cDNA Synthesis Service

BOC Sciences provides cDNA synthesis services in which the synthesized first-strand cDNA can be widely used for second-strand cDNA synthesis for PCR amplification, hybridization, cDNA library creation, and full-length cDNA synthesis.

• First-strand cDNA Synthesis
  First-strand cDNA synthesis utilizes oligo (dT), random primers, or a combination of these strategies to initiate the reverse transcription reaction. Initiation of the reaction with oligo(dT) preferentially starts at the 3' end of the RNA fragment.
• Second-strand cDNA Synthesis
  Methods for synthesizing the second-strand of cDNA can be selected depending on the needs and application. The methods we currently offer include the following.

• Analysis of cDNA
  The cDNA synthesis product needs to be validated and analyzed to ensure that the customer gets the target product.

cDNA Synthesis Steps

• Sample Preparation
  RNA is the template for cDNA synthesis, and RNA needs to maintain its integrity during the extraction process.
• Removal of Genomic DNA
  Before cDNA synthesis, DNase I must be completely removed because the residual enzyme degrades single-stranded DNA. Double-stranded specific DNase can be used as an alternative to DNase I to remove contaminating gDNA without affecting RNA or single-stranded DNA.
• Selecting a Reverse Transcriptase
  Depending on the need and application, select the appropriate reverse transcriptase to ensure efficient cDNA synthesis.
• Formulating the Reaction System
  In addition to the enzyme and primers, the main reaction components for reverse transcription include an RNA template (pre-treated to remove genomic DNA), buffer, dNTP, DTT, RNAase inhibitor and nuclease-free water.
• cDNA Synthesis
  cDNA synthesis includes cDNA first and second-strand synthesis.

Materials to be Provided by the Customer

• Total RNA: volume ≥10μL, concentration ≥50ng/μL
• Cell sample: 106 cells or more, cell suspension or cell precipitation
• Tissue sample: volume of tissue ≥100mg

Reference

1. Meis J E, et al. RNA amplification and cDNA synthesis for qRT-PCR directly from a single cell[J]. 2009.