What is cDNA?
Complementary DNA (cDNA) is DNA synthesized from an mRNA template via the enzyme reverse transcriptase. It represents only the expressed genes of a genome (i.e., exons only, no introns).
Steps in cDNA Synthesis
RNA Extraction
Isolate high-quality total RNA or mRNA from your sample (e.g., tissue, cells).
DNase Treatment (Optional)
Treat RNA with DNase to remove contaminating genomic DNA.
Primer Selection
You can use one of the following primers:
Oligo(dT): Binds to the poly-A tail of mRNA (eukaryotic-specific). Oligo(dT) primers, which are 12–20 deoxythymidine sequences, offer specific annealing to the poly(A) tails of eukaryotic mRNAs. This makes them highly effective for cDNA library construction. However, their reliance on intact poly(A) tails means they are not appropriate for degraded RNA.
Random hexamers: Bind randomly to all RNA, including rRNA and tRNA.In contrast to the poly(A)-tail specific oligo(dT) primers, random primers (typically hexamers of random deoxyribonucleotides, [d(N)6]) can prime cDNA synthesis from a wider range of mRNAs, regardless of the presence of a poly(A) tail. Furthermore, their non-specific nature allows them to be used for DNA synthesis with Klenow fragments on DNA templates.
Gene-specific primers: For targeted reverse transcription.
Reverse Transcription Reaction
Combine RNA, primers, dNTPs, reverse transcriptase enzyme, RNase inhibitor, and buffer.
Incubate at appropriate temperatures (typically 42–55°C for 30–60 min depending on the enzyme).
cDNA Storage or Use
The cDNA can be stored at –20°C or used directly in PCR, qPCR, or cloning.
A typical protocol for cDNA synthesis (Protoscript II First Strand cDNA Synthesis Kit):
If denaturation of template RNA is desired, use the following protocol.
1. Mix RNA sample and primer d(T)23VN or random hexamer in a sterile RNase-free microfuge tube.
COMPONENT VOLUME
Total RNA: 1–6 μl (up to 1 μg)
Primer: d(T)23VN or random hexamers: 2 μl
Nuclease-free H2O to a total volume of 8 μl
2. Denature sample RNA/ primer d(T)23 VN / random hexer for 5 minutes at 65°C. Spin briefly and put promptly on ice.
3. Add reaction mix and enzymes:
ProtoScript II Reaction Mix (2X): 10 μl
ProtoScript II Enzyme Mix (10X): 2 μl
IF using random hexamer mix, perform an initial 25oC incubation for 5min before the cDNA step.
4. Incubate the 20 μl cDNA synthesis reaction at 42°C for 1 hour (60min).
5. Inactivate the (RT) enzyme at 80°C for 5 minutes.
6. The cDNA product should be stored at –20°C.
Common Reverse Transcriptase Enzymes
M-MLV (Moloney Murine Leukemia Virus RT)
AMV (Avian Myeloblastosis Virus RT)
Superscript II/III/IV (modified M-MLV with reduced RNase H activity for better yield)
Watch our video on how cDNA is typically prepared in the lab.
cDNA Applications
1. Quantitative Gene Expression Analysis (qPCR)
cDNA serves as the template for quantitative real-time PCR (qPCR), enabling precise measurement of transcript abundance. It avoids RNA instability and allows for specific amplification using DNA polymerases.
2. RNA-Seq and Transcriptome Profiling
High-throughput RNA sequencing (RNA-seq) begins with cDNA synthesis from RNA. This allows next-generation sequencing platforms to capture the transcriptome, revealing gene expression patterns, splice variants, fusion transcripts, and noncoding RNAs.
3. Molecular Cloning of Eukaryotic Genes
Since eukaryotic mRNA is spliced, using cDNA bypasses introns. Genes expressed in prokaryotic systems (e.g., E. coli) must be intron-free; hence, cDNA provides a proper template for heterologous expression of functional proteins.
4. Alternative Splicing Analysis
By generating cDNA and amplifying specific gene regions, researchers can identify and validate alternative splicing events, transcript isoforms, and post-transcriptional modifications.
5. Functional Genomics and Gene Discovery
cDNA libraries (collections of cDNA from specific tissues or developmental stages) enable gene identification, full-length transcript capture, and expression profiling. This is essential for gene annotation, cDNA microarrays, and characterizing unknown genes.
6. Single-Cell Transcriptomics
In single-cell RNA-seq (scRNA-seq), cDNA synthesis is crucial after capturing RNA from individual cells, enabling ultra-sensitive quantification of cell-to-cell variation in gene expression.
7. CRISPR Guide RNA Design and Validation
To target expressed genes or validate transcript-level knockdowns (e.g., siRNA, shRNA), researchers use cDNA to confirm whether a transcript is present and accessible for guide design.
8. In Vitro Transcription Templates
cDNA can be cloned downstream of a promoter (e.g., T7) to synthesize RNA probes or synthetic RNA via in vitro transcription, used in hybridization assays or functional RNA studies.References
Thermo Fisher Scientific. (n.d.). Real-Time PCR Basics. https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics.html
Excedr. (2022, August 31). cDNA Synthesis: Overview & Applications. https://www.excedr.com/resources/cdna-synthesis-overview
Haddad, F. (2010). Reverse transcription of the ribonucleic acid: the first step in RT-PCR assay. Methods in molecular biology (Clifton, N.J.), 630, 129–136.
New England Biolabs. (n.d.). RNases. https://www.neb.com/en/products/rna-synthesis-and-modification/rnases
Thermo Fisher Scientific. (n.d.). Five Steps to Fast RT-PCR. https://www.thermofisher.com/mx/en/home/life-science/pcr/reverse-transcription/superscript-iv-one-step-rt-pcr-system/5-steps-rtpcr.html
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