METHODS FOR RNA QUANTIFICATION

• US 20180371536A1
• Filed: 05/26/2016
• Published: 12/27/2018
• Est. Priority Date: 06/01/2015
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fu, Glenn, Wilhelmy, Julie
• Granted Patent: US 11,124,823 B2
• Time in Patent Office: Days
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The disclosure provides for methods, compositions, systems, devices, and kits for quantification of RNA, and determination of efficiency of re verse transcription of RNA to cDNA.

Claims

1. A method for determining the functional integrity of an RNA sample, comprising:

hybridizing a plurality of oligonucleotides comprising a target specific region that specifically binds to a target in an mRNA molecule of one or more reference genes in the RNA sample, wherein at least 10% of the plurality of oligonucleotides comprises different molecular labels;

extending the plurality of oligonucleotides to generate a plurality of nucleic acid molecules of the one or more reference genes;

counting the number of molecular labels associated with the plurality of nucleic acid molecules; and

determining the functional integrity of the RNA sample based on the number of molecular labels associated with the one or more reference genes.

2. The method of claim 1, wherein at least one of the one or more reference genes is a housekeeping gene.

6. The method of claim 1, wherein the RNA sample comprises total RNA.

7. The method of claim 1, wherein the RNA sample comprises mRNA.

8. The method of claim 1, wherein the RNA sample comprises partially degraded RNA.

9. The method of claim 1, wherein the extending step comprises reverse transcription of the mRNA molecule.

10. The method of claim 1, wherein each of the plurality of oligonucleotides comprises a binding site for a primer, and comprising amplifying the plurality of nucleic acid molecules using a primer that binds to the binding site.

12. The method of claim 10, wherein each of the plurality of oligonucleotides or the primer comprises an optically detectable label.

13. The method of claim 12, wherein the optically detectable label is a fluorescent label.

14. The method of claim 12, wherein the plurality of oligonucleotides comprises different fluorescent labels for each of the one or more reference genes.

15. The method of claim 1, wherein counting the number of molecular labels associated with the plurality of nucleic acid molecules comprises determining the sequences of the plurality of nucleic acid molecules by sequencing.

16. The method of claim 1, wherein counting the number of molecular labels associated with the plurality of nucleic acid molecules comprises hybridizing the plurality of nucleic acid molecules to a microarray comprising a plurality of probes that specifically binds to the molecular labels of the plurality of oligonucleotides.

18. The method of claim 1, comprising:

adding a plurality of spike-in RNA molecules to the RNA sample, wherein at least 10% of the plurality of spike-in RNA molecules comprises different molecular labels;

hybridizing a reverse-transcription oligonucleotide to the plurality of spike-in RNA molecules;

extending the reverse-transcription oligonucleotide to generate a second plurality of nucleic acid molecules;

counting the number of molecular labels associated with the second plurality of nucleic acid molecules; and

determining the efficiency of reverse-transcription based on the ratio of the number of molecular labels associated with the second plurality of nucleic acid molecules to the number of molecular labels of the plurality of spike-in RNA molecules.

19. The method of claim 18, wherein counting the number of molecular labels associated with the second plurality of nucleic acid molecules comprises determining the sequences of the second plurality of nucleic acid molecules by sequencing.

20. The method of claim 18, wherein counting the number of molecular labels associated with the second plurality of nucleic acid molecules comprises hybridizing the second plurality of nucleic acid molecules to a microarray.

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29. A method for determining efficiency of reverse transcription of an RNA sample, comprising:

adding a plurality of spike-in RNA molecules to the RNA sample, wherein at least 10% of the plurality of spike-in RNA molecules comprises different molecular labels;

hybridizing an reverse-transcription oligonucleotide to the plurality of spike-in RNA molecules;

extending the reverse-transcription oligonucleotide to generate a plurality of nucleic acid molecules;

counting the number of molecular labels associated with the plurality of nucleic acid molecules; and

determining the efficiency of reverse-transcription based on the ratio of the number of molecular labels associated with the plurality of nucleic acid molecules to the number of molecular labels of the plurality of spike-in RNA molecules.

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37. A kit for determining transcription efficiency of an RNA sample, comprising:

a plurality of spike-in RNA molecules, wherein at least 10% of the plurality of spike-in RNA molecules comprises different molecular labels; and

a reverse-transcription oligonucleotide that specifically binds to the plurality of spike-in RNA molecules.

44. The kit of claim 37, wherein the plurality of spike-in RNA molecules comprises 960 different molecular labels.

45. The kit of claim 37, wherein the plurality of spike-in RNA molecules each comprises a two restriction sites flanking the molecular label.

47. The kit of claim 37, further comprising a plurality of oligonucleotides that specifically binds to mRNA molecules of a reference gene.

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