Scientific

qPCR Calculator

Calculating Differential Gene Expression through qPCR

This free RTPCR/qPCR Calculator takes as input a tab separated file (CSV) and calculates the up and down-regulation of genes between two samples using the ΔΔ-CT method, as explained in this lecture.

What is reported ?

The calculation comprises the following steps:
  1. cleaning up the input data, thereby removing all CP/CT values that are non-numerical (E.g, empty entries, bracketed entries [21], or cycles larger than 40).
  2. estimating the qPCR/RTPCR efficiencies for each Gene/Cell combination based on all measured dilutions and normalizing all CP/CT values to an efficiency of 100%
  3. calculate the Δ-CT values from each gene to all listed household genes.
  4. calculate the ΔΔ-CT values between all cell types (e.g: between Normal and siRNA)
  5. report on the up-/down-regulation ratios and on the efficiencies of the qPCR.

An example of such a report is given below. In this experiment, a large number of groups have been rejected due to very uncertain dilution series. The remaining groups have been reported in the Up/Down regulations table.

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Up/down regulations

Household Gene From To Direction Ratio Log(ratio)
GADPH TPM4 siRNA Normal Up 1.37929 0.463925
GADPH TAF9 siRNA Normal Down 3.54528 -1.8259
GADPH TAF1B siRNA Normal Up 1.72315 0.78505
GADPH TAF15 siRNA Normal Up 3.97223 1.98995
GADPH NRG1 siRNA Normal Up 9.31496 3.21955
GADPH MBTPS2 siRNA Normal Up 2.44575 1.29028
GADPH HIF1A siRNA Normal Down 1.35973 -0.443325
GADPH BCHE siRNA Normal Down 1.20226 -0.26575
BCHE TPM4 siRNA Normal Up 1.65827 0.729675
BCHE TAF9 siRNA Normal Down 2.94885 -1.56015
BCHE TAF1B siRNA Normal Up 2.07168 1.0508
BCHE TAF15 siRNA Normal Up 4.77566 2.2557
BCHE NRG1 siRNA Normal Up 11.199 3.4853
BCHE MBTPS2 siRNA Normal Up 2.94043 1.55602
BCHE HIF1A siRNA Normal Down 1.13098 -0.177575
BCHE GADPH siRNA Normal Up 1.20226 0.26575

Efficiencies

Cell Gene Average Efficiency Comment
Normal BCHE 105.841 Accepted group
Normal FOS 170.712 Rejected group
Normal GABARAP 121.973 Rejected group
Normal GADPH 91.5267 Accepted group
Normal GTF2B 133.654 Rejected group
Normal HIF1A 92.6202 Accepted group
Normal LZIC nan Rejected group
Normal MBTPS2 95.3834 Accepted group
siRNA BCHE 111.697 Accepted group
siRNA FOS 191.324 Rejected group
siRNA GABARAP 146.561 Rejected group
siRNA GADPH 97.6546 Accepted group
... ...
 ... ...
siRNA MBTPS2 93.8257 Accepted group
siRNA NEFH 326.86 Rejected group
siRNA NMU 70.5202 Rejected group
siRNA NRG1 97.0566 Accepted group
siRNA TAF15 110.147 Accepted group
siRNA TAF1B 89.7493 Accepted group
siRNA TAF6 129.888 Rejected group
siRNA TAF9 97.1325 Accepted group
siRNA TPM4 111.765 Accepted group
The qPCR Up/Down Ratio Calculator - http://analysis.yellowcouch.org/qpcr/