Methods for isolating long fragment rna from fixed samples

A long-segment, tissue-sample technology that is used in the determination of chemotherapy-based regimens and can address issues such as failure

Inactive Publication Date: 2010-07-21
RESPONSE GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As a result, other researchers have made consistent, but unsuccessful attempts to obtain oligonucleotide primer sets that allow quantification of DPD expression in paraffinized tissues

Method used

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  • Methods for isolating long fragment rna from fixed samples
  • Methods for isolating long fragment rna from fixed samples
  • Methods for isolating long fragment rna from fixed samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209] Example 1: Extraction steps of long fragment RNA

[0210] I. Tissue Preparation

[0211] Paraffin blocks containing 10 micron sections of FFPE tissue were mounted on glass slides without coverslips using standard protocols. For deparaffinization and nuclear fast red (NFR) staining, slides were processed as follows:

[0212] Slides were washed twice with xylene for 5 minutes each, followed by ethanol ("EtoH") washes. The slides were stained with NFR using standard protocol.

[0213] The area of ​​interest (eg, tumor tissue or stroma) is dissected by hand or with a laser capture microdissector (depending on the size of the resection).

[0214] II. RNA extraction

[0215] An extraction solution containing Tris / HCL, EDTA, SDS and water was prepared. Add tumor tissue and proteinase K to the extraction solution in the centrifuge tube. The sample is then heated at an appropriate temperature for an appropriate time in order to obtain a maximum yield of long fragment RNA, ...

Embodiment 2

[0218] Example 2: Method for Determining the Length Distribution of Isolated RNA

[0219] To determine the relative amounts of various RNA fragment lengths isolated from FFPE tissues, the following strategy was used. RNA isolated from FFPE samples using the present invention and other known extraction methods is converted to cDNA using oligo dT primers. This means that only mRNA fragments containing the 3'-oligo-A tail will be extended and converted into cDNA, providing a starting point for measuring fragment length. PCR amplification of β-actin mRNA was used to represent the total mRNA population. Primers were chosen to amplify a segment of approximately 100-120 bp representing the β-actin gene at positions 100, 300, 400 and 1000 bp from the 3'-end of the mRNA (Figure 2). Using this strategy, any differences in the length-dependent efficacy of the amplification were minimized, rather than actually trying to amplify 100, 300, 400 and 1000 bp fragments. Therefore, the Ct of ...

Embodiment 3

[0222] Example 3: Effect of Proteinase K

[0223] This example demonstrates the effect of proteinase K concentration on RNA yield and DNA contamination. Proteinase K concentration was varied over a 4-fold range (5-20 μg, 1X-4X as indicated in the graph) at 50°C with incubation times of 0.5, 2, 3 and 16 hours. like Figure 4As shown, 1X (5 μg) of proteinase K gave a greater amount of approximately 2-fold (1 Ct) RNA yield, however more importantly, amounts of proteinase K greater than 1X gave visible higher DNA contamination (2 -3Ct cycle). This experiment also demonstrates the effect of incubation time on the amount of DNA extracted, with a 16 hour incubation time being 3 to 7 Ct cycles better than shorter incubation times. DNA in the extracts was detected by performing PCR without first performing a reverse transcription reaction to convert the RNA to cDNA ("no reverse transcription or NRT control"). This way, only PCR amplification of the co-extracted DNA occurs, if it is...

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Abstract

The present invention relates to methods for the extraction of long fragment RNA from fixed tissue specimens. In particular, the present invention relates to methods for the extraction of RNA from formalin-fixed paraffin-embedded tissue specimens for use in biologic applications, including assays based on oligonucleotide hybridization.

Description

[0001] This application claims priority to Provisional Application No. 60 / 945,785, filed June 22, 2007, which is hereby incorporated by reference in its entirety. technical field [0002] The present invention relates to the field of extraction and isolation of high yield and high quality (long fragment) RNA from fixed tissue samples. The present invention relates to the use of these novel extraction methods to provide a method for assessing gene expression levels of genes (eg, cancer biomarkers) in fixed or fixed and paraffin-embedded tissues. The present invention also provides methods for determining a chemotherapy-based regimen by measuring the mRNA level of a biomarker in tumor cells of a patient and comparing it to a predetermined threshold expression level. Background technique [0003] Quantitative measurement of RNA species (RNA species) is the core issue in the modern research of molecular biology. RNA species are also of great clinical importance, for example, in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1003C12Q1/6806C12Q1/6886
Inventor 凯瑟琳·达南伯格
Owner RESPONSE GENETICS
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