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US 2006/0234270 A1Published application
Normalization methods for gene expression analysis
AssigneeAffymetrix, Inc.
InventorThomas Houts
Life of this patent
Estimating the term…
Abstract
The invention provides methods for obtaining an expected concentration for each of a plurality of housekeeping genes and using these values to obtain normalized concentrations for genes in an experimental sample using both external transcripts and internal housekeeping genes. A standard calibration curve is prepared using intensity values and concentrations of external transcripts. The standard calibration curve is used to obtain an expected concentration for each housekeeping gene. A calibration curve in an experimental sample is used to obtain preliminary concentrations for housekeeping genes and a correction factor is calculated from the expected concentrations. The correction factor is used to normalize values for genes in the experimental sample.
Claims31
A method for obtaining an expected concentration for each of a plurality of housekeeping genes comprising:
- (a) obtaining a nucleic acid sample obtained from a biological sample;
- (b) obtaining a set of external transcripts wherein said set comprises a plurality of different external transcripts each present at different known concentrations;
- (c) adding an aliquot of said external transcripts to said biological sample to generate a spiked sample;
- (d) amplifying said spiked sample to generate an amplified sample;
- (e) labeling said amplified sample to generate a labeled sample;
- (f) hybridizing said labeled sample to an array to obtain a hybridization pattern;
- (g) analyzing said hybridization pattern to obtain an intensity value for each external transcript and for each of said housekeeping genes;
- (h) preparing a standard calibration curve with said intensity values of said external transcripts on a first axis and concentrations of said external transcripts on a second axis;
- (i) fitting a curve through a plurality of points of said standard calibration curve;
- (j) plotting an intensity value for each housekeeping gene onto said standard calibration curve;
- (k) calculating an estimated concentration for each housekeeping gene from said standard calibration curve;
- (l) repeating steps (a) through (k) for a plurality of biological samples;
- and
- (m) combining said estimated concentrations for each housekeeping gene to obtain an expected concentration for each housekeeping gene.
11 more elements
A method for obtaining a normalized concentration for a target in an experimental sample comprising:
- (a) obtaining a nucleic acid sample obtained from said experimental sample;
- (b) obtaining a set of external transcripts wherein said set comprises a plurality of different external transcripts each present at different concentrations within a range of concentrations;
- (c) adding an aliquot of said external transcripts to said experimental sample to generate a spiked sample;
- (d) amplifying said spiked sample to generate an amplified sample;
- (e) labeling said amplified sample to generate a labeled sample;
- (f) hybridizing said labeled sample to an array to obtain a hybridization pattern;
- (g) analyzing said hybridization pattern to obtain an intensity value for each external transcript and for each of said housekeeping genes;
- (h) preparing an experimental calibration curve with said intensity values of said external transcripts on a first axis and concentrations of said external transcripts on a second axis;
- (i) fitting a line through a plurality of points of said experimental calibration curve;
- (j) plotting said intensity values from each housekeeping gene onto said experimental calibration curve;
- (k) calculating a preliminary concentration for each said housekeeping gene;
- (l) determining a ratio of expected concentration of said housekeeping gene from said experimental sample to said preliminary concentration for each housekeeping gene and averaging a plurality of said ratios to obtain a correction factor;
- (m) plotting said intensity value for said target onto said second calibration curve to obtain a preliminary concentration for said target in said experimental sample;
- and
- (n) multiplying said preliminary concentration for said target by said correction factor to obtain a normalized concentration for said target in said experimental sample.
12 more elements
A computer software product for obtaining a normalized concentration for a target in an experimental sample comprising:
- (a) computer program code for preparing an experimental calibration curve with intensity values of external transcripts on a first axis and concentrations of said external transcripts on a second axis;
- (b) computer program code for fitting a line through a plurality of points of said experimental calibration curve;
- (c) computer program code for plotting said intensity values from each housekeeping gene onto said experimental calibration curve;
- (d) computer program code for calculating a preliminary concentration for each said housekeeping gene;
- (e) computer program code for determining a ratio of expected concentration of said housekeeping gene from said experimental sample to said preliminary concentration for each housekeeping gene and averaging a plurality of said ratios to obtain a correction factor;
- (f) computer program code for plotting said intensity value for said target onto said second calibration curve to obtain a preliminary concentration for said target in said experimental sample;
- and
- (g) computer program code for multiplying said preliminary concentration for said target by said correction factor to obtain a normalized concentration for said target in said experimental sample.
5 more elements
A computer readable medium having computer executable instructions for obtaining a normalized concentration for a target in an experimental sample comprising instructions for:
- (a) preparing an experimental calibration curve with intensity values of external transcripts on a first axis and concentrations of said external transcripts on a second axis;
- (b) fitting a line through a plurality of points of said experimental calibration curve;
- (c) fitting said intensity values from each housekeeping gene to said experimental calibration curve;
- (d) calculating a preliminary concentration for each said housekeeping gene;
- (e) determining a ratio of expected concentration of said housekeeping gene from said experimental sample to said preliminary concentration for each housekeeping gene and averaging a plurality of said ratios to obtain a correction factor;
- (f) plotting said intensity value for said target onto said second calibration curve to obtain a preliminary concentration for said target in said experimental sample;
- and
- (g) multiplying said preliminary concentration for said target by said correction factor to obtain a normalized concentration for said target in said experimental sample.
5 more elements
Dependencies are inferred from the claim text.