IP Library › Granted Patent US 9,150,909
Granted Patent B2
US 9,150,909 · App. 12/548,226 · Granted Oct 6, 2015

Determination of the integrity of RNA

Inventor: Vladimir Denisov (Santa Cruz, CA)
Assignee: Bio-Rad Laboratories, Inc.
C12Q1/6816C12Q1/6806C12Q2545/113C12Q2563/107C12Q2565/125G01N2333/4728G01N2800/34
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Quick Facts
Patent No.
US 9,150,909
App. No.
12/548,226
Granted
Oct 6, 2015
Kind
B2
Abstract

Methods, systems, and apparatus make a determination of a level of integrity of a sample of biomolecules. For example, the determination of the integrity of RNA in a sample may be done in a fast and reproducible manner, such that the user can be assured of accuracy of a test (e.g. quantitative polymerase chain reaction qPCR) on the sample and compare results of different samples. The determination of integrity of an RNA sample is performed by comparing a size profile to reference size profiles (degradation standards) obtained from degradation over different lengths of times. As the reference scale of the level of integrity is derived from the actual degradation that occurs in a sample, high accuracy, reproducibility, and efficiency is provided.

Claims (58)

1. A method of improving accuracy and reproducibility of determining a level of integrity of a sample of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) molecules, the method comprising:

receiving a first size profile of the sample of the RNA and/or DNA molecules, wherein a size profile provides a measure of a distribution of values of at least one dimension of the RNA and/or DNA molecules, in the sample, the at least one dimension including length and/or weight;

comparing, with an electrophoresis system, the first size profile to a plurality of reference size profiles, wherein each reference size profile correlates to a different level of integrity; and

based on a similarity of the first size profile to one or more of the reference size profiles, determining, with the electrophoresis system, the level of integrity of the sample of RNA and/or DNA molecules,

wherein the plurality of reference size profiles is obtained by:

at each of a plurality of different elapsed times relative to an initial time:

measuring a respective reference size profile of a reference sample of RNA and/or DNA molecules selected from a group of reference samples having about the same initial integrity, wherein each respective reference size profile corresponds to a different amount of degradation of the reference sample, wherein each reference size profile provides a measure of a distribution of values of the at least one dimension of the RNA and/or DNA molecules in the reference sample; and

mapping each reference size profile to a level of integrity, a highest integrity level of reference size profile being measured at the initial time, and each successive lower integrity level of reference size profile being measured at a longer elapsed time from the initial time.

2. The method of claim 1 , wherein the reference size profiles are derived from one or more reference samples containing RNA and/or DNA molecules of the same origin and the same concentration.

3. The method of claim 1 , wherein the level of integrity is expressed as a numerical value within predetermined scale.

4. The method of claim 1 , wherein the different elapsed times of degradation of the reference size profiles are zero and successive periodic times.

5. The method of claim 1 , wherein the reference size profiles map linearly to different levels of integrity.

6. The method of claim 1 , wherein a size profile comprises a plurality of ratios of at least three regions, wherein each region corresponds to a different amount of time for RNA and/or DNA molecules of the sample to reach a detection point, wherein RNA and/or DNA molecules of different size reach the detection point at different times.

7. The method of claim 6 , wherein the comparing includes:

comparing one or more size features of the first size profile to corresponding size features of the reference size profiles,

wherein determining the level of integrity of the sample of RNA and/or DNA molecules includes:

based on the comparing, determining an integrity value for each size feature of the first size profile; and

averaging the integrity values to determine the level of integrity of the sample of RNA and/or DNA molecules.

8. The method of claim 7 , wherein the one or more size features include a ratio of areas of a size profile around the rRNA peaks of 18S and 28S.

9. The method of claim 7 , wherein averaging weights one size feature higher if the integrity value of the one size feature is higher than a threshold value.

10. The method of claim 7 , wherein for each size feature of the first size profile:

comparing one or more size features of the first size profile to corresponding size features of the reference size profiles includes:

identifying two reference profiles having corresponding size features with values between which lies the value for the respective size feature of the first size profile; and

calculating the differences between the respective size feature of the first size profile and the values of the corresponding size features of the two reference profiles, and

determining an integrity value includes:

interpolating between the values of the corresponding size features of the two reference size profiles to determine the integrity value of the respective size feature.

11. The method of claim 1 , wherein the size profile is an electropherogram.

12. A computer program product comprising a non-transitory computer readable medium storing a plurality of instructions for controlling a processor to perform an operation for of determining a level of integrity of a sample of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) molecules, the instructions comprising:

receiving a first size profile of the sample of the RNA and/or DNA molecules, wherein a size profile provides a measure of a distribution of values of at least one dimension of the RNA and/or DNA molecules in the sample, the at least one dimension including length and/or weight;

comparing the first size profile to a plurality of reference size profiles, wherein each reference size profile correlates to a different level of integrity; and

based on a similarity of the first size profile to one or more of the reference size profiles, determining the level of integrity of the sample of RNA and/or DNA molecules,

wherein the plurality of reference size profiles is obtained by:

at each of a plurality of different elapsed times relative to an initial time:

measuring a respective reference size profile of a reference sample of RNA and/or DNA molecules selected from a group of reference samples having about the same initial integrity, wherein each respective reference size profile corresponds to a different amount of degradation of the reference sample, wherein each reference size profile provides a measure of a distribution of values of the at least one dimension of the RNA and/or DNA molecules in the reference sample; and

mapping each reference size profile to a level of integrity, a highest integrity level of reference size profile being measured at the initial time, and each successive lower integrity level of reference size profile being measured at a longer elapsed time from the initial time.

13. An electrophoresis system comprising:

a voltage source;

a detector;

the computer program product of claim 12 ; and

one or more processors communicably coupled with the detector and the computer program product.

14. The electrophoresis system of claim 13 , wherein the computer program product stores the reference size profiles.

15. The electrophoresis system of claim 13 , further comprising a light source that excites luminescent markers on the biomolecules, the light source illuminating a first electromagnetic radiation at a detection point, and wherein the detector detects a second electromagnetic radiation emitted from the excited biomolecules at the detection point at a specific instance in time.

16. A method of deriving reference size profiles for an electrophoresis system, the method comprising:

receiving a reference sample of biomolecules;

at each of a plurality of different elapsed times relative to an initial time:

measuring, with an electrophoresis system, a respective size profile of the reference sample of biomolecules, wherein a size profile provides a measure of a distribution of values of at least one dimension of the biomolecules in the reference sample,

wherein each respective size profile corresponds to a different amount of degradation of the reference sample; and

mapping each size profile to a level of integrity, wherein a size profile measured at a later elapsed time maps to a lower level of integrity.

17. The method of claim 16 , further comprising:

storing, in a computer readable medium of the electrophoresis system, the size profiles associated with the corresponding level of integrity.

18. The method of claim 16 , wherein the reference sample has no degradation prior to the measurement of a first size profile at the initial time.

19. The method of claim 16 , wherein the mapping is non-linear.

20. The method of claim 16 , further comprising:

degrading the reference sample at the plurality of elapsed times by subjecting the reference sample to heat.

21. The method of claim 17 , further comprising:

repeating for one or more additional reference samples, each selected from a group of reference samples having about the same initial integrity; and

taking an average of the size profiles taken at each elapsed time, wherein the average of the size profiles is stored in the computer readable medium.

22. The method of claim 16 , wherein the plurality of different elapsed times are predetermined, and wherein a respective size profile is measured when a predetermined elapsed times is reached.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 6, 2010
From: DENISOV, VLADIMIR
To: BIO-RAD LABORATORIES, INC.
Reel/Frame 024805/0161 →
Continuity (2)
Provisional Application 61093060 · Aug 29, 2008
Related Publication 20100057371A1 · Mar 4, 2010