What is cDNA?
Complementary DNA (cDNA) is DNA synthesized from an mRNA template via the enzyme reverse transcriptase. It represents the expressed genes of a genome (i.e., exons only, no introns). cDNA is made artificially in the lab from mRNA and represents only the expressed genes at the time the RNA was collected.
This is in contrast with the genome, which includes all genetic material in the cell, whether coding or non-coding, introns, promoters, repetitive sequences, etc.
When preparing cDNA, depending on your end goal, you may want what is referred to as the first strand, which is single-stranded DNA. When intending to do cloning, a single strand of DNA is not useful: you need double-stranded DNA.
Steps in cDNA Synthesis
RNA Extraction
Isolate high-quality total RNA or mRNA from your sample (e.g., tissue, cells).
DNase Treatment (Optional but highly recommended)
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.
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)
Example Protocol: Maxima H Minus Double Stranded cDNA Synthesis Kit
STEPS
First Strand cDNA synthesis:
Mix RNA + primer (1ul) + water up to 14 µL.
Heat at 65°C for 5 min, then chill on ice.
Add:
5 µL 4X First Strand Reaction Mix
1 µL First Strand Enzyme Mix
Final vol. is 20ul
For oligo(dT) or gene-specific primers: Incubate at 50°C for 30 min.
For random hexamers: Incubate 25oC for 10 min, then 50oC for 30 min..
Inactivate enzyme: 85°C for 5 min, then chill on ice.
Second Strand cDNA Synthesis
20 µL First strand cDNA
55 µL Water
20 µL 5X Second Strand Mix
5 µL Second Strand Enzyme Mix
Final vol. is 100ul
Incubate at 16°C for 60 min.
Stop reaction: Add 6 µL of 0.5 M EDTA (pH 8).
Residual RNA Removal
Add 10 µL RNase I (10 U/µL)
Incubate 5 min at room temp
Purify your double-stranded cDNA (blunt end) using:
Column-based PCR Purification Kit, or
Phenol:chloroform extraction
Tip: Elute in ≤ 20 µL buffer for higher cDNA concentration.
🎥 Want to See It in Action?
Check out our video tutorial on cDNA synthesis on the Adwoa Biotech YouTube Channel, where we walk through the process.
Subscribe by Email
Follow Updates Articles from This Blog via Email
No Comments