Normalization of data

• US 8,089,623 B2
• Filed: 08/18/2009
• Issued: 01/03/2012
• Est. Priority Date: 09/19/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Kao, H. Pin, Harding, Ian A., Karger, Achim, Oldham, Mark F., Ostadan, Omead, Young, Greg
• Primary Examiner(s): Evans, F. L.
• Application Number:
  US12/543,459
• Publication Number:
  US 20100220321A1
• Time in Patent Office: 868 Days
• Field of Search: None
• US Class Current: 356/243.1
• CPC Class Codes: B01L 2200/021 Adjust spacings in an array of wells, pipettes or holders, format transfer between arrays of different size or geometry
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2300/022 Transponder chips
• CPC Class Codes: B01L 2300/044 pierceable, e.g. films, membranes
• CPC Class Codes: B01L 2300/046 Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0293 for liquids
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: B01L 3/563 Joints or fittings in general F16L; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 9/50 : Clamping means, tongs in general F16B2/06
• CPC Class Codes: B01L 9/523 : for multisample carriers, e.g. used for microtitration plates
• CPC Class Codes: G01N 2035/00366 : Several different temperatures used
• CPC Class Codes: G01N 21/274 : Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 35/028 : having reaction cells in the form of microtitration plates
• CPC Class Codes: H04N 25/63 : applied to dark current
• CPC Class Codes: H04N 25/68 : applied to defects
• CPC Class Codes: Y10T 436/10 : Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

Methods for normalizing output from an instrument employing a reference standard or non-fluorescing substance disposed within at least one of a plurality of reaction chambers. The method comprises collecting and analyzing a signal associated with the reference standard or non-fluorescing substance to determine a normalizing bias. The normalizing bias is then applied to the data signal collected from a remainder of the plurality of reaction chambers.

Claims

1. A method of normalizing spectral sensitivity from an instrument, the instrument using a microplate having a plurality of reaction chambers, the instrument having a plurality of filters, the method comprising:

testing a first reference standard deposited in a first reaction chamber of the plurality of reaction chambers in the microplate, wherein the first reference standard is operable for outputting a first reference standard signal in response to the testing;

testing a sample aliquot deposited in a second reaction chamber of the plurality of reaction chambers, wherein the sample aliquot includes at least a portion of a polynucleotide sample, at least one dye is deposited in the second reaction chamber, and the at least one dye is operable for outputting a data signal representative of the polynucleotide sample in response to the testing;

detecting the first reference standard signal and the data signal;

generating a normalization bias based on analyzing the first reference standard signal relative to a predetermined level; and

normalizing the data signal relative to the normalization bias.

2. The method according to claim 1, further comprising:

depositing a second reference standard into a third reaction chamber of the plurality of reaction chambers in the microplate, wherein the second reference standard is operable for outputting a second reference standard signal.

3. The method according to claim 2, further comprising:

detecting the second reference standard signal, wherein the second reference standard signal is analyzed with the first reference standard signal relative to a predetermined level to generate the normalization bias.

4. The method according to claim 1 wherein the at least one dye comprises a series dilution.

5. The method according to claim 1 wherein the at least one dye comprises multiple titration points.

6. The method according to claim 1 wherein the at least one dye comprises dye ladders.

7. The method according to claim 1 further comprising performing reverse transcription on the sample aliquot.

8. A method of normalizing data output from an instrument, the instrument using a microplate having a plurality of reaction chambers, wherein a first reaction chamber of the plurality of reaction chambers is free from a fluorescing substance, the method comprising:

depositing a sample aliquot and at least one dye into a second of the plurality of reaction chambers, the sample aliquot having at least a portion of the polynucleotide sample, wherein the dye is operable for outputting a data signal in response to the polynucleotide sample;

determining a level of background correction based on a signal from the first reaction chamber of the plurality of reaction chambers;

generating a normalization bias based on the determined level of background correction; and

normalizing the data signal relative to the normalization bias.

9. The method according to claim 8, further comprising:

depositing a non-template control in the first reaction chamber of the plurality of reaction chambers.

10. The method according to claim 8 wherein the at least one dye comprises a series dilution.

11. The method according to claim 8 wherein the at least one dye comprises multiple titration points.

12. The method according to claim 8 wherein the at least one dye comprises dye ladders.

13. A method of normalizing data output from an instrument, the instrument using a microplate having a plurality of reaction chambers, the method comprising:

detecting a first control signal output from a first control in a first reaction chamber of the plurality of reaction chambers, wherein the first reaction chamber is free of a polynucleotide sample;

detecting a data signal output from at least one dye, wherein a sample aliquot and the at least one dye are in a second reaction chamber of the plurality of reaction chambers, the sample aliquot including at least a portion of the polynucleotide sample, and the at least one dye is operable for outputting the data signal in response to the polynucleotide sample;

generating a normalization bias based on analyzing the first control signal relative to a predetermined level; and

normalizing the data signal relative to the normalization bias.

14. The method according to claim 13,wherein the first control is a non-template control.

15. The method according to claim 13 wherein the at least one dye comprises a series dilution.

16. The method according to claim 13 wherein the at least one dye comprises multiple titration points.

17. The method according to claim 13 wherein the at least one dye comprises dye ladders.