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Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference

An internal reference, plasma technology, applied in the fields of genetic engineering and oncology, can solve the problems of restricting clinical translation and unable to control biological differences, etc.

Active Publication Date: 2012-05-09
南京江北新区生物医药公共服务平台有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although the above two methods can effectively control the experimental differences, they cannot control the biological differences of the individual itself. The lack of specific and stable serum / plasma miRNA internal reference still restricts the clinical transformation of serum / plasma miRNA research results and the inter-laboratory research. Comparison of Research Results

Method used

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  • Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference
  • Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference
  • Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference

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Experimental program
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Embodiment 1

[0069] The collection of embodiment 1 sample and the arrangement of sample data

[0070] The inventor has collected a large amount of serum / plasma samples of various tumor patients and healthy controls from multiple tertiary first-class hospitals in Jiangsu Province since July 2003, and the cases and control samples used for research are collected at the same period, and sampling, analysis The installation and storage conditions were uniform, and by sorting out the sample data, the inventor selected 354 samples that met the following criteria as experimental samples for Solexa sequencing, TLDA chip detection, and a series of subsequent qRT-PCR verifications:

[0071] 1. Cancer patients diagnosed by pathology, including cases of lung cancer, breast cancer, gastric cancer, liver cancer and cervical cancer in the internal reference screening stage, and esophageal cancer, colon cancer, rectal cancer, pancreatic cancer, oral cancer, gastric cancer in the internal reference verificat...

Embodiment 2

[0075] Example 2 Solexa sequencing experiment of miRNA in serum / plasma

[0076] The above qualified samples include 60 cases of lung cancer (30 cases of long survival group and 30 cases of short survival group), 48 cases of breast cancer (24 cases / group), 40 cases of gastric cancer (20 cases / group), 30 cases of liver cancer, 30 cases of cervical cancer and 48 healthy male controls, 48 ​​healthy female controls. These 10 groups of people were subjected to the Solexa sequencing test (the kit was purchased from ABI Company) to obtain relevant results. The specific steps are:

[0077] 1. Take 50ml of serum / plasma from each group of samples, and add an equal volume of Trizol reagent;

[0078] 2. Phase separation: place at room temperature for 15 minutes, then add chloroform according to the volume ratio of 0.2ml chloroform / 1ml Trizol reagent, shake for 15s, room temperature for 15 minutes, centrifuge at 12,000g, 4°C for 15 minutes;

[0079] 3. Transfer the aqueous phase to a new...

Embodiment 3

[0089] TLDA chip detection of miRNA in embodiment 3 serum / plasma

[0090] The above 10 groups of samples subjected to Solexa sequencing were detected by TLDA chip (the kit was purchased from ABI Company) to obtain relevant results. The specific steps are:

[0091] 1. Take 600 μl of serum / plasma from each group and add 3 times the volume of Trizol reagent;

[0092] 2. Phase separation: place at room temperature for 15 minutes, add cel-miR-39 (TAKARA) with a final concentration of 10-4pmol / μl as an internal reference, then add chloroform equal to the volume of serum / plasma, shake for 50s, room temperature for 15 minutes, 14,000rpm, Centrifuge at 4°C for 15 minutes;

[0093] 3. RNA precipitation: transfer the water phase to a new 15ml centrifuge tube, add 1.5 times the volume of the water phase in absolute ethanol, and mix well;

[0094] 4. Enrich RNA with QIAGEN miRNeasy kit: pipette 700 μl of sample into the spin column each time, centrifuge at 14,000 rpm for 15 s, discard t...

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Abstract

The invention belongs to the fields of genetic engineering and oncology and discloses an internal reference for detecting miRNA (micro Ribonucleic Acid) in serum / blood plasma and application of the internal reference. The internal reference is separate miR-484 or miR-191 or a combination of the miR-191 and the miR-484. The internal reference and a primer of the internal reference can be used for preparing an internal reference detection kit which is used for detecting the internal reference of the miRNA in serum / blood plasma.

Description

field of invention [0001] The invention belongs to the fields of genetic engineering and oncology, and relates to an internal reference for serum / plasma miRNA detection and application thereof. Background technique [0002] MicroRNAs (ie miRNAs) is a hotspot in the field of tumor molecular biology research in recent years. Mature miRNA is a kind of small molecule non-coding RNA with a length of about 21-24 nucleotides. In the nucleus, miRNA is first expressed as the original (primary miRNA, pri-miRNA) with a length of hundreds of nucleotides, and then expressed by Drosha Nucleases are processed into about 100 nucleotide-long precursors (precursor miRNA, pre-miRNA), which are transported to the cytoplasm through an Exportin-5-dependent mechanism; in the cytoplasm, they are further processed into mature miRNAs by Dicer nuclease. Mature miRNAs mainly regulate gene expression through complementary base pairing with the 3'-untranslated region (UTR) of target mRNAs, which can lea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/113C12N15/11
Inventor 沈洪兵胡志斌张辰宇董静
Owner 南京江北新区生物医药公共服务平台有限公司
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