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Molecular markers, internal reference genes and application thereof, detection kit and method for constructing detection model

A technology of molecular markers and internal reference genes, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as expensive, genes and their detection models that have yet to be verified, and achieve reduction Effects of over-treatment and inappropriate treatment, satisfying individualized precise treatment, and perfecting technical methods

Active Publication Date: 2018-01-12
CAPITALBIO CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these products are currently developed based on European and American populations. After these products enter China, they are not only expensive, but whether the genes and their detection models are suitable for Chinese populations has yet to be verified.

Method used

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  • Molecular markers, internal reference genes and application thereof, detection kit and method for constructing detection model
  • Molecular markers, internal reference genes and application thereof, detection kit and method for constructing detection model
  • Molecular markers, internal reference genes and application thereof, detection kit and method for constructing detection model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The collection of embodiment 1 sample, the arrangement of sample data

[0063] Using FFPE samples from newly diagnosed breast cancer patients without treatment, systematically collected complete clinical follow-up data, and through sorting out the sample data, the inventor selected 341 samples that met the following criteria as TLDA (Taqman LowDensity Array, TLDA) chip detection And a series of experimental samples verified by qRT-PCR:

[0064] (1) newly diagnosed patients with untreated early and mid-stage breast cancer;

[0065] (2) Patients with untreated early, mid-stage ER or (and) PR-positive breast cancer;

[0066] (3) LN has or (and) has not been transferred, and the amount of LN transferred;

[0067] (4) Accurate and detailed follow-up information is available.

Embodiment 2

[0068] Example 2 TLDA chip screening molecular markers and internal reference genes

[0069] TLDA chip detection was performed on 26 breast cancer samples with good prognosis and 26 breast cancer samples with poor prognosis that met the above conditions, and relevant results were obtained. The specific steps are:

[0070] (1) RNA extraction from FFPE samples: 4 slices of 20 μm slices or 8 slices of 10 μm slices were taken from each sample, and RNA was extracted according to the instruction of HighPure FFPET RNA Isolation Kit (Roche), and the extracted RNA was quantitatively controlled by NanoDrop-2000 Afterwards, downstream reverse transcription experiments were performed.

[0071] (2) cDNA sample was obtained by reverse transcription reaction of total RNA: take 1 μg total RNA according to VILO TM Master Mix kit (Invitrogen) manual was used for reverse transcription.

[0072] (3) TLDA detection of cDNA samples: the above cDNA products and After the Universal PCR MasterM...

Embodiment 3

[0077] Example 3 Large sample size qRT-PCR verification of molecular markers

[0078] 14 molecular markers and 5 internal reference genes screened by TLDA: BCL2, PGR, SCUBE2, ESR1, MKi67, CCNB1, MYBL2, GRB7, ERBB2, MMP11, CD68, BAG1, MAPT, MS4A1; ACTB, GAPDH, GUSB, NUP214 , VCAN. A single-tube qRT-PCR verification was performed using 289 FFPE samples that met the above sample collection requirements and complete clinical follow-up information.

[0079] (1) RNA extraction of 289 cases of FFPE samples: 4 slices of 20 μm slices or 8 slices of 10 μm slices were taken from each sample, and RNA was extracted according to the instruction of HighPure FFPET RNA Isolation Kit (Roche). The extracted RNA was quantified and qualified by NanoDrop-2000 After the control, the downstream reverse transcription experiment was carried out.

[0080] (2) 289 cases of FFPE sample RNA were reverse-transcribed into cDNA: take 1 μg of total RNA according to VILO TM MasterMix kit (Invitrogen) manu...

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Abstract

The invention especially relates to molecular markers, internal reference genes and application thereof, a detection kit and a method for constructing a detection model, belonging to the field of biotechnology. With follow-up information used for comparison, the kit provided by the invention has accuracy of 70% in predication of the risk of recurrence or death of first-visit patients with ER-positive and PR-positive breast cancers in 3-10 years after operations, wherein the prediction accuracies of a low-risk group and a high-risk group are 81.1% and 54.4%, respectively while the prediction accuracies of corresponding FFPE pathological examination results are 71.9 to 56.8%, respectively. The risk prediction model matching with the kit only needs the Ct values of the molecular markers, theage of a patient, the stage of pT and the number of LN and does not depend on other clinical pathological information, so the risk prediction model has better performance in evaluation of prognosis ofbreast cancers than simple pathological prediction results, can reduce malpractice caused by errors in pathological prediction and further improves a technical method for evaluation of prognosis of breast cancers.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a molecular marker, an internal reference gene and its application, a detection kit and a method for constructing a detection model. Background technique [0002] Breast cancer is one of the main reasons that threaten the life and health of women all over the world. In 2013, the American Cancer Society released national cancer statistics showing that the incidence of breast cancer ranks first among female cancers, and its mortality rate ranks second. According to the latest data from the National Cancer Center, there were 232,340 new breast cancer cases and 39,620 deaths among American women in 2013. In the United States, one in eight women develops breast cancer. Although China is a country with a low incidence of breast cancer, its incidence and mortality have increased significantly in recent years. Of the 1.3 million newly diagnosed breast cancer patients worldwide each year, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12N15/11C12Q1/68
Inventor 郭弘妍孙义民王亚辉谢展邢婉丽程京邓涛张治位
Owner CAPITALBIO CORP
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