118,078,431,372.55
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118,078,431,372.55
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The Transcript Copy Number Calculator converts a known mass of RNA into the number of transcript molecules present in the sample. This is essential for preparing RT-qPCR absolute quantification standards, calibrating in vitro transcribed RNA standards, and estimating gene expression levels in absolute terms. Enter the RNA amount in nanograms and the transcript length in nucleotides to calculate the number of copies using Avogadro's number.
The transcript copy number is calculated using:
Copies = (RNA amount in ng × 6.022 × 10²³) / (Transcript length in nt × 340 × 10⁹)
Where:
The formula derives from: moles = mass / MW, and copies = moles × Avogadro's number.
Inputs
Results
100 ng of a 1500-nt transcript contains approximately 1.18 × 10¹¹ copies, suitable for making serial dilution standards for qPCR.
Inputs
Results
10 ng of a short 500-nt transcript yields approximately 3.54 × 10¹⁰ copies.
First, in vitro transcribe your target gene to generate a pure RNA standard of known concentration. Use this calculator to determine the copy number of your stock. Then make 10-fold serial dilutions from 10⁸ down to 10¹ copies per µL. Use 1–2 µL of each dilution as template in your qPCR standard curve to enable absolute quantification of your unknown samples.
RNA standards are preferred because they undergo the reverse transcription step along with your unknown samples, controlling for RT efficiency variation. DNA standards bypass the RT step and may overestimate target abundance if RT efficiency is low. However, DNA standards are more stable and easier to prepare, so many labs use them with appropriate correction factors.
For in vitro transcribed standards, use the actual length of your transcript including any vector-derived sequences. For endogenous mRNA, include the full transcript length from 5' cap to poly(A) tail. The poly(A) tail (typically 200–250 nt) contributes to the molecular weight and should be included for accurate copy number calculation.
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