DNA-meter, a calibrated microarray
For each experiment, organize microarray signal intensities into a simple text file of two columns, ProbeID Intensity:
A_67_P05181479 184.6739
A_67_P20444947 300.5581
A_67_P04577152 6376.69
A_53_P127264 37.15217
... ...
Run average.exe (download | .cpp) to average signal intensities per probe, assign experiment ID and merge experiments into one file and calculate standard errors.
average file1.txt id1 file2.txt id2... > out.txt
id1, id2, etc are experiment IDs; for calibration use concentrations.
A calibration curve produced from a dilution series experiment (example dataset, output).
average calib_0_0625.txt 0.0625 calib_1.txt 1 calib_2.txt 2 calib_0_5.txt 0.5 calib_0_25.txt 0.25 calib_0_125.txt 0.125 > calibration_curve.txt
Single experiment (exp1_1_1raw.txt output)
average exp1_1_1raw.txt 1_1_1 > exp1_1_1avg.txt
If the microarray is already calibrated, proceed to Step 3.
Run calibrate.exe (download | .cpp), which will use merged averaged signal intensities of the calibration curve and will produce parameters of calibration. Calibration output format is ProbeID a b R2 CurveID:
A_53_P100398 0.000691116 0.00325183 0.997242 L
A_53_P100736 4.28337 0.296658 0.981139 F
A_53_P102504 0.000593089 0.00524655 0.998836 L
... ...
calibrate < calibration_curve_file.txt [-T] [-R2 {0.9}] [-freund {F}] [-linear {LN}] [-langmuir {LG}] > out.txt
Input file produced in step 1.
-T tabulated output for inspection elsewhere
-R2 {0.9} R2 cut off, e.g 0.9, provided by user
-freund {F} Freundlich calibration; output marker, e.g. F, provided by user
-linear {LN} Linear calibration; output marker, e.g. LN, provided by user
-langmuir {LG} Langmuir calibration; output marker, e.g. LG, provided by user
When more than one calibration curve suggested, the one with the highest R2 is saved into the output.
Input file is produced by Step 1 (calibration_curve.txt, output)
calibrate < calibration_curve.txt -R2 0.98 -freund F -langmuir L > calibration.txt
Run concentration.exe (download | .cpp) using averaged signal intensities from Step 1 and calibration parameters from Step 2 to calculate concentrations and standard errors.
concentration < calibrated_probes_file.txt [-freund {F}] [-linear {LN}] [-langmuir {LG}] -intens sample1.txt sample2.txt... > out.txt
-freund {F} Freundlich calibration y=exp(a)*x^b; input marker, e.g. F, provided by user
-linear {LN} Linear calibration y = a+b*x; input marker, e.g. LN, provided by user
-langmuir {LG} Langmuir calibration y = Y_max*K*x/(1+K*x); input marker, e.g. LG, provided by user a = 1/(K*Y_max); b = 1/Y_max
Averaged signal intensities for an experiment are produced in Step 1 (exp1_1_1avg.txt) and calibration parameters are produced in Step 2 (calibration.txt). Intensities are calculated and saved into output.
concentration < calibration.txt –freund F -langmuir L -intens exp1_1_1avg.txt > conc_1_1_1.txt