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.
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 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.
| Method | Specifications |
| Self-priming method | The 3' end of the synthesized single-stranded cDNA is able to form a short hairpin structure, which provides a ready-made primer for the synthesis of the second strand. The second strand of the cDNA is synthesized by using the E. coli DNA polymerase I Klenow fragment or reverse transcriptase after denaturation of the DNA-RNA hybridization strand from the product of the first-stranded synthesis reaction, and the loop is digested with S1 nuclease specific for the single strand, which can be further cloned. |
| Replacement synthesis method | In the presence of dNTP, RNAase H is utilized to create cuts and nicks in the mRNA strand of the hybridized strand. This results in a series of RNA primers for the synthesis of the second strand, which is synthesized under the action of E. coli DNA polymerase I. |
| Analysis method | Specifications |
| Gel Electrophoresis | The size and purity of the cDNA can be initially determined by the electrophoretic migration rate. |
| Real-time quantitative PCR (qPCR) | qPCR measures the expression level of a target gene and provides relative or absolute quantitative results. |
| Sequence Analysis | Sanger sequencing or high-throughput sequencing technology enables cDNA sequence analysis. |
cDNA (complementary DNA) is synthesized from RNA through the process of reverse transcription using RNA as a template. The resulting cDNA is complementary to the RNA sequence and can be used for downstream applications like PCR, hybridization, and gene expression analysis.
The main steps in cDNA synthesis are RNA preparation, removal of genomic DNA, selection of reverse transcriptase, and synthesis of both the first and second strands of cDNA. The second strand can be synthesized using various methods like self-priming or replacement synthesis.
For first-strand cDNA synthesis, oligo(dT) primers, random primers, or a combination of both are used to initiate the reverse transcription reaction. Oligo(dT) primers preferentially bind to the 3' end of mRNA, while random primers provide broader coverage for diverse RNA species.
To ensure high-quality cDNA, it is essential to use intact RNA, properly remove genomic DNA, and select the appropriate reverse transcriptase enzyme. After synthesis, the cDNA can be analyzed using gel electrophoresis, qPCR, or sequencing for validation.
Second-strand cDNA synthesis can be performed using the self-priming method or replacement synthesis method. The self-priming method utilizes a hairpin structure formed at the 3' end of the first-strand cDNA, while the replacement synthesis method uses RNAase H to generate primers from the mRNA strand.
At BOC Sciences, we ensure high-quality cDNA by using specialized enzymes, maintaining strict quality control during the synthesis process, and validating the final product through gel electrophoresis, qPCR, and sequencing.
To initiate cDNA synthesis, you need to provide RNA (≥10μL, ≥50ng/μL), or cell/tissue samples (≥106 cells or ≥100mg tissue). Proper sample preparation is essential for obtaining accurate and high-quality cDNA.
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