Deep Science Publishing
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Chapter 26
Synthesis of complementary DNA (C-DNA): Principles,
methodology, and applications in gene expression studies
Yashdeep Srivastava1*, Keshawanand Tripathi1, Narendra Kumar2
1
Department of Biotechnology, Invertis University, Bareilly, Uttar Pradesh, India.
2
School of Biotechnology and Bioengineering, Institute of Advanced Research, Gandhinagar,
Gujarat, India.
*
Email: yashbubiotech@[Link]
1. Introduction:
Complementary DNA (cDNA) synthesis from RNA is a pivotal technique in molecular
biology that enables the conversion of RNA molecules into DNA copies (Gubler, and
Hoffman, 1983). This process holds immense significance in various research fields,
including gene expression analysis, functional genomics, and molecular cloning. By
synthesizing cDNA from RNA templates, researchers can access a stable DNA form of
RNA sequences, allowing for easier manipulation, amplification, and analysis (Green,
and Sambrook, 2012). The synthesis of cDNA from RNA involves the reverse
transcription (RT) of RNA molecules using a specialized enzyme called reverse
transcriptase (Okayama, and Berg, 1982). This enzyme catalyzes the synthesis of DNA
strands complementary to the RNA template. Through this process, the genetic
information contained within RNA molecules can be preserved and further analyzed
using various molecular techniques commonly applied to DNA (Srivastava et al., 2022).
cDNA synthesis plays a crucial role in numerous experimental procedures, such as gene
expression profiling using techniques like quantitative reverse transcription PCR (qRT-
PCR) and RNA sequencing (RNA-seq) (Brown, 2010; Tripathi et al 2013a,b).
Additionally, cDNA libraries constructed from specific tissues, cells, or organisms serve
as valuable resources for studying gene expression patterns, identifying novel
transcripts, and understanding cellular processes at the molecular level.
2. Materials required:
1. Total RNA sample
2. Oligo(dT) primer
3. RNase-free water
4. RNase inhibitor
5. Reverse transcriptase enzyme
6. Nucleotide mix (dNTPs)
7. Reaction buffer
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8. RNase inhibitor
9. Thermal cycler
10. PCR tubes or plates
11. Microcentrifuge tubes
12. Pipettes and tips
13. Ice bucket
3. Procedure:
1. Prepare a master mix for cDNA synthesis by combining the following components
in a microcentrifuge tube on ice:
Table 1: Master mix
Component Volume (μL)
Oligo(dT) primer 1
Total RNA sample X (as per instructions)
RNase inhibitor 1
RNase-free water Adjust to 10
2. Thoroughly blend the master mix by gently vortexing and briefly centrifuging.
3. Subject the master mix to a thermal cycler, incubating it at 65°C for 5 minutes to
initiate RNA and primer denaturation.
4. Promptly transfer the tube to an ice bath for 2 minutes to induce cooling.
5. Create a reverse transcription reaction mix by incorporating the following
components into the master mix:
Table 2: Add on components in master mix
Component Volume (μL)
5X Reaction Buffer 4
RNase Inhibitor 1
Reverse Transcriptase 1
Nucleotide Mix (dNTPs) 2
RNase-Free Water Adjust to 20
6. Thoroughly blend the reaction mix by pipetting up and down gently.
7. Place the reaction mix in a thermal cycler and incubate it at 42°C for 60-90 minutes
to facilitate reverse transcription.
8. Inactivate the reverse transcriptase enzyme by heating the reaction mix at 70°C for
10 minutes.
9. Utilize the cDNA promptly for PCR amplification or store it at -20°C for future
applications.
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4. PCR Amplification of cDNA (Optional):
1. Prepare a PCR reaction mixture by combining the following components in a PCR
tube or plate (10 μL) :
Table 3: PCR reaction mixture
Component Volume (μL)
cDNA template 1
Forward and Reverse Primers 2
Taq DNA Polymerase 1
PCR Buffer 4
dNTPs 0.5
RNase-Free Water 1.5
2. Modify the volume to achieve the desired reaction volume.
3. Thoroughly blend the PCR reaction mixture by gently pipetting up and down.
4. Position the PCR tubes or plate in a thermal cycler and execute the PCR program
as per the assay's specifications.
5. Assess the PCR products through agarose gel electrophoresis or an alternative
suitable method for analysis.
5. Precautions:
1. Ensure all equipment and surfaces are clean and RNase-free to prevent RNA
degradation.
2. Use RNase inhibitors and wear gloves to minimize RNA degradation during
handling.
3. Work quickly and efficiently to minimize RNA exposure to RNases.
4. Use a suitable reverse transcriptase enzyme and buffer according to manufacturer's
instructions.
5. Ensure RNA samples are of high quality and integrity before starting cDNA
synthesis.
6. Perform a genomic DNA elimination step if necessary to prevent contamination
with genomic DNA.
7. Maintain consistent and appropriate incubation temperatures during the cDNA
synthesis reaction.
8. Perform a negative control reaction without reverse transcriptase to detect
genomic DNA contamination.
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9. Use random primers or gene-specific primers based on the application and
experimental design.
10. Verify the quality and quantity of synthesized cDNA using gel electrophoresis or
qPCR.
11. Store synthesized cDNA at -20°C or -80°C to maintain stability and integrity.
12. Dispose of unused reagents and contaminated materials properly according to
laboratory waste disposal guidelines.
6. References:
1. Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual
(3rd ed.). Cold Spring Harbor Laboratory Press.
2. Brown, T. A. (2010). Gene cloning and DNA analysis: An introduction (6th ed.).
Wiley-Blackwell.
3. Wilson, K., & Walker, J. (2010). Principles and techniques of biochemistry and
molecular biology (7th ed.). Cambridge University Press.
4. Green, M. R., & Sambrook, J. (2012). Molecular cloning: A laboratory manual
(4th ed.). Cold Spring Harbor Laboratory Press.
5. Gubler, U., & Hoffman, B. J. (1983). A simple and very efficient method for
generating cDNA libraries. Gene, 25(2–3), 263–269. [Link]
6. Okayama, H., & Berg, P. (1982). High-efficiency cloning of full-length cDNA.
Molecular and Cellular Biology, 2(2), 161–170. [Link]
7. Tripathi, K., Sharma, N. K., Rai, V., & Rai, A. K. (2013). Low cellular P-quota
and poor metabolic adaptations of the freshwater cyanobacterium Anabaena
fertilissima Rao during Pi-limitation. Antonie van Leeuwenhoek, 103, 277–291.
8. Tripathi, K., Sharma, N. K., Kageyama, H., Takabe, T., & Rai, A. K. (2013).
Physiological, biochemical, and molecular responses of the halophilic
cyanobacterium Aphanothece halophytica to Pi-deficiency. European Journal of
Phycology, 48(4), 461–473.
9. Srivastava, Y., Tripathi, S., Mishra, B., & Sangwan, N. S. (2022). Cloning and
homologous characterization of geranylgeranyl pyrophosphate synthase (GGPPS)
from Withania somnifera revealed alterations in metabolic flux towards
gibberellic acid biosynthesis. Planta, 256(1), 4.
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