Materials and methods for quality-controlled two-color RT-QPCR diagnostic testing of formalin fixed embedded and/or fresh-frozen samples

Patent metadata

Field	Value
Publication number	US-9528161-B2
Application number	US-201514619285-A
Country	US
Kind code	B2
Filing date	Feb 11, 2015
Priority date	Feb 12, 2014
Publication date	Dec 27, 2016
Grant date	Dec 27, 2016

Abstract

Described herein are materials, methods, and kits enabling accurate and reproducible two-color reverse-transcription real-time quantitative PCR (RT-qPCR) for quality-controlled molecular diagnostic testing of samples that may contain degraded RNA. In certain aspects described herein are materials, methods, and kits for use in the molecular diagnostic testing of lung cancer in FFPE samples and/or fresh-frozen samples. Also described herein are materials and methods to control for inter-experimental variation occurring during two-color RT-qPCR amplification arising from variation in fluorescence specific activity, use of different thermocyclers, and inter-laboratory differences.

First claim

What is claimed is:

1. A kit for use in molecular diagnostic testing of lung cancer in a sample, comprising:
a) synthetic competitive internal standards (IS), individual IS comprising one each of: SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; or SEQ ID NO: 24, wherein known quantities of each IS are formulated into an internal standards mixture (ISM);
b) an external standards mixture (ESM) comprising:
(1) purified synthetic native template (NT), individual NT comprising one each of: SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO:23; and
(2) synthetic competitive internal standards (IS), individual IS comprising one each of: SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; or SEQ ID NO: 24,
wherein known quantities of each purified synthetic NT and IS are formulated into the ESM at a ratio in the range of 10:1 to 1:10;
c) pairs of gene-specific primers (GSP) specific to genes ACTB (forward primer: SEQ ID NO: 1; reverse primer: SEQ ID NO: 2), CDKN1A (forward primer: SEQ ID NO: 3; reverse primer: SEQ ID NO: 4), MYC (forward primer: SEQ ID NO: 5; reverse primer: SEQ ID NO: 6), and E2F1 (forward primer: SEQ ID NO: 7; reverse primer: SEQ ID NO: 8); and
d) sequence-specific fluorometric hydrolysis probes, individual fluorometric hydrolysis probes comprising one each of: SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; or SEQ ID NO: 16; wherein all fluorometric hydrolysis probes are labeled with a fluorescent reporter.

2. The kit according to claim 1 , wherein SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15 are labeled with FAM, and SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 are labeled with Quasar 670.

3. The kit according to claim 1 , wherein the known quantities of each purified synthetic NT and IS are formulated into the ESM at a ratio of 1:1.

4. A method for using the kit according to claim 1 comprising the steps of:
a) extracting RNA from the sample;
b) reverse-transcribing (RT) the RNA extracted from the sample;
c) pre-amplifying cDNA generated by the RT of the RNA extracted from the sample along with the ISM, wherein the pre-amplification of the cDNA and ISM is done by polymerase chain reaction (PCR) using the pairs of GSP specific to the genes ACTB, CDKN1A, MYC, and E2F1;
d) performing a second round of PCR amplification of genes ACTB, CDKN1A, MYC, and E2F1, wherein a reaction mixture for a single gene comprises:
(1) a diluted pre-amplified cDNA and ISM product;
(2) a primer mixture comprising pairs of GSP corresponding to genes ACTB, CDKN1A, MYC, and/or E2F1;
(3) fluorometric hydrolysis probes specific for the single gene NT and corresponding IS;

e) along with the second round of PCR amplification simultaneously performing PCR amplification of each individual gene in reaction mixtures containing two distinct concentrations of the ESM, and primers and fluorometric probes for each individual gene NT and IS, wherein each distinct PCR reaction comprises the same fluorometric hydrolysis probes specific for the single gene NT and corresponding IS of step d)(3); and
f) quantifying the copy number for loading control gene ACTB and target genes CDKN1A, MYC, and E2F1.

5. The method of claim 4 , wherein, along with the second round of PCR amplification, the method further includes:
simultaneously performing PCR amplification of genes ACTB, CDKN1A, MYC, and E2F, wherein a reaction mixture for a single gene comprises:
(1) a first concentration of the ESM;
(2) the primer mixture comprising the pairs of GSP corresponding to genes ACTB, CDKN1A, MYC, and E2F1; and
(3) fluorometric hydrolysis probes specific for the single gene NT and corresponding IS; and,

repeating step e) using a second concentration of the ESM in place of the first concentration of the ESM, wherein the first concentration of the ESM and the second concentration of the ESM are distinct.

6. The method according to claim 4 , wherein the two distinct concentrations of ESM differ by at least one order of magnitude.

7. The method according to claim 4 , wherein the two distinct concentrations of ESM are 10 −13 M NT/10 −13 M IS and 10 −14 M NT/10 −14 M IS.

8. The method according to claim 4 , wherein the step f) of quantifying the copy number for each target gene comprises the steps of:
a) calculating the difference in quantification cycle (Cq) between the NT and IS for a target gene using the formula [NT Cq−IS Cq] Target Gene ;
b) calculating an average difference in Cq between the NT and IS of two concentrations of ESM using the formula ([NT Cq−IS Cq] ESM Concentration 1 +[NT Cq−IS Cq] ESM Concentration 2 )/2=[NT Cq−IS Cq] ESM ;
c) calculating a corrected delta Cq using the formula [NT Cq−IS Cq] Target Gene −[NT Cq−IS Cq] ESM ;
d) obtaining a target gene NT copy number by multiplying 2 (−corrected delta Cq) by a known number of input IS copies corresponding to the target gene in the reaction; and
e) normalizing the target gene NT copy number to an ACTB gene loading control NT value.

9. The method of claim 4 , wherein the samples comprise material having degraded RNA therein.

10. The method of claim 4 , wherein the samples comprise formalin fixed paraffin embedded (FFPE) samples.

11. The method of claim 4 , wherein the samples comprise fresh-frozen samples.

12. A kit for use in molecular diagnostic testing of a sample, comprising:
e) one or more synthetic competitive internal standards (IS), wherein each IS is synthesized to correspond with a unique target gene or reference gene, wherein an IS probe binding site has a 4-6 bp difference than that of a native template (NT), and wherein known quantities of the one or more IS are formulated into an internal standards mixture (ISM);
f) an external standards mixture (ESM) comprising:
(1) one or more purified synthetic NT, wherein each NT is synthesized to correspond with a unique target gene or reference gene; and
(2) one or more synthetic competitive internal standards (IS), wherein each IS corresponds with a unique target gene or reference gene,
wherein known quantities of each purified synthetic NT and IS are formulated into the ESM at a ratio in the range of 10:1 to 1:10;
g) one or more pairs of gene-specific primers (GSP), wherein each pair of GSP is specific for a unique target gene or reference gene, and is designed to amplify a PCR product having a product size of approximately 60-80 base pairs and span introns/exon splice junctions of the unique target gene or reference gene;
h) at least one pair of fluorometric hydrolysis probes, wherein each pair of fluorometric probes is specific for a unique target gene or reference gene, and comprises a first fluorometric hydrolysis probe that is sequence-specific for an NT probe binding site and is labeled with a first fluorescent reporter, and a second fluorometric hydrolysis probe that is sequence specific for an IS probe binding site of an IS corresponding to the NT against which the first fluorometric hydrolysis probe is sequence-specific for, and is labeled with a second fluorescent reporter.

13. The kit of claim 12 , wherein in step b) 2), known quantities of each purified synthetic NT and IS are formulated into the ESM.

14. The kit of claim 13 , wherein in step b) 2), known quantities of each purified synthetic NT and IS are formulated into the ESM at a ratio of 1:1.

15. The kit of claim 12 , wherein in step b) 2), one or more synthetic competitive internal standards (IS) corresponds to each of the one or more purified synthetic NT.

16. The kit of claim 12 , wherein the first fluorometric hydrolysis probe seq

Assignees

• Univ Toledo

Inventors

• Willey James C
• Yeo Jiyoun
• Crawford Erin

Classifications

• G06F19/24

  Physics · mapped topic

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title