Don’t Know How Much cDNA to Use? Still Measuring cDNA Concentration?

Quantitative PCR (qPCR) is a widely used technique in molecular biology, often perceived as straightforward. However, successful qPCR experiments hinge on numerous subtle yet crucial factors. One of the most overlooked aspects is the cDNA template—the quality and quantity of cDNA directly determine experimental success. Poor cDNA preparation leads to failed qPCR runs. In this article, we demystify cDNA use in qPCR and help you clear this first critical hurdle. (Yes, qPCR has many more technical pitfalls—stay tuned for future posts!)

How to Determine the Right Amount of cDNA

1. Can you measure cDNA concentration with a Nanodrop?
Not recommended. Reverse transcription products are complex mixtures containing untranscribed RNA, enzymes, cDNA, and various salts or ions. UV absorbance methods like Nanodrop cannot distinguish between RNA and DNA and are easily affected by protein and salt contaminants. As a result, the measured concentration is inaccurate and unsuitable for downstream qPCR quantification.

2. Can gel electrophoresis assess cDNA quality?
Barely. Since total RNA contains transcripts of all sizes, cDNA products appear as smears on agarose gels. Plus, reverse transcription doesn't amplify material like PCR does, so the overall cDNA yield is low. Bands may be faint or even invisible.

(Example: cDNA smear pattern from gel electrophoresis — image source: internet)

 3. How to determine optimal cDNA dilution?
Run a dilution series of your cDNA and perform qPCR on each. Select the dilution where Ct values fall between 15–30 cycles—this is the ideal range.
Alternatively, start with undiluted cDNA and adjust based on the initial Ct value. Since Ct is logarithmically related to template amount (ΔCt of 1 = 2× concentration difference), you can back-calculate the appropriate dilution.
Example: Ct = 12 is too low; to get Ct = 16, dilute 2⁴ = 16×.

4. What if the target gene and reference gene show vastly different Ct values?
If Ct values differ significantly despite using the same dilution, it suggests large differences in gene expression. You may use different dilutions for target and reference genes to ensure both fall within the 15–30 Ct range. Just make sure that within each gene, dilutions remain consistent across all experimental groups.

How to Obtain High-Quality cDNA

1. RNA Extraction and Storage
Use fresh samples flash-frozen in liquid nitrogen or preserved in RNA-stabilizing reagent. Store at -80°C and avoid repeated freeze-thaw cycles.
Extract RNA using TRIzol or a commercial kit. Work in an RNase-free environment with appropriate PPE and certified RNase-free consumables.
Dissolve RNA in RNase-free water and store short-term at -20°C or long-term at -80°C.
Use UV absorbance to assess purity (A260/280 ≥ 2.0; A260/230 ≈ 1.8–2.1). However, this method doesn’t assess RNA integrity. Gel electrophoresis is recommended to check for degradation and contaminants.

 

(A high-quality RNA gel shows clear 5S, 18S, and 28S bands; the 28S band should be twice the intensity of 18S.)

2. Remove Genomic DNA Contamination
Genomic DNA (gDNA) contamination can lead to non-specific amplification or abnormally low Ct values. Use intron-spanning primers and either DNA-removal extraction kits or reverse transcription kits with gDNA removal steps.

3. Choose the Right Reverse Transcription Primers

Primer Type	Features	Pros	Cons	Combination method
Oligo(dT)	12–20 T bases, binds poly-A tail	Full-length cDNA synthesis	Only for polyadenylated mRNA; sensitive to template quality
Random Primers	6–9 bases, bind randomly	Suitable for complex/low-abundance templates	Low specificity, shorter fragments
Gene-Specific Primers (GSPs)	Bind a specific RNA sequence	High sensitivity and specificity	Only generate target-specific cDNA

For mRNA qPCR, a mix of oligo(dT) and random primers is commonly recommended.

Related products

The products provided by Yeasen are as follows.

Table 1. Related products