500
ng
0.5
µg
500
ng
0.5
µg
The RNA-to-DNA Conversion Calculator estimates the expected cDNA yield from a reverse transcription (RT) reaction based on the input RNA amount and conversion efficiency. Reverse transcription is the first step in RT-PCR and RNA-seq workflows, converting messenger RNA into complementary DNA for amplification and analysis. Understanding the expected yield helps in planning downstream experiments and troubleshooting failed reactions.
The expected cDNA yield is calculated as:
cDNA Yield (ng) = RNA Input (ng) × Conversion Efficiency / 100
Key considerations:
Inputs
Results
Starting with 1000 ng (1 µg) of total RNA and 50% conversion efficiency, you can expect approximately 500 ng of cDNA.
Inputs
Results
2 µg RNA with a high-efficiency enzyme (75%) yields approximately 1500 ng (1.5 µg) of cDNA.
Most commercial RT kits achieve 40–60% conversion efficiency under optimal conditions. This means starting with 1 µg of total RNA, you can expect 400–600 ng of cDNA. High-performance enzymes like SuperScript IV or Maxima H Minus can achieve up to 80%. Factors that reduce efficiency include RNA degradation, secondary structure, presence of inhibitors, and suboptimal reaction temperature.
For RT-qPCR, typical inputs are 100 ng to 2 µg of total RNA per reaction. For abundant targets, 100–500 ng is usually sufficient. For rare transcripts, use 1–2 µg. Ensure all samples in a comparative experiment use the same amount of input RNA for valid relative quantification.
This calculator works with total RNA input. Note that mRNA comprises only about 1–5% of total cellular RNA, with the rest being ribosomal RNA (rRNA) and transfer RNA (tRNA). If you are using oligo(dT) priming, only the poly(A)+ mRNA fraction is reverse-transcribed. Random hexamer priming will reverse-transcribe all RNA species.
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