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Universal amplification of fragmented RNA

a technology of rna and amplification method, which is applied in the field of universal amplification of rna, can solve the problems of ineffective step, insufficient material for analysis without prior amplification of rna, etc., and achieve the effect of improving the sensitivity of rt-pcr and improving the sensitivity of rna analysis methods

Inactive Publication Date: 2005-09-08
GENOMIC HEALTH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] The present invention provides a sample preparation method that enables global amplification of even very small or very fragmented RNA samples. The method of the invention improve the sensitivity of RNA analysis methods, including RT-PCR and hybridization arrays. Furthermore, the methods of the invention permit the measurement of mRNA levels of all expressed genes including fragmented and / or blocked mRNA species in archived paraffin-embedded tissue samples. This method also permits the measurement of non-polyadenylated mRNA such as histones and non-coding RNAs, such as microRNAs (miRNAs). The invention may additionally include an enhanced reverse transcription step and a modified PCR step that increases the sensitivity of RT-PCR used for gene expression profiling of fragmented RNA samples.

Problems solved by technology

In many situations where gene expression profiling is potentially useful, there is insufficient material for analysis without prior amplification of RNA.
This step, however, is not effective if the source of mRNA is a fixed, paraffin-embedded tissue sample, which may have been archived as long as 10-20 years, and in which the RNA is badly degraded (Lewis et al., J. Pathol. 195:66-71 (2001)).

Method used

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specific embodiments

[0094] Three representative protocols (A, B and C) of the invention are illustrated in FIG. 1. Processes A and B start with FPET RNA and involve 1) direct or indirect unblocking of the 3′ OH on the terminal nucleotide, 2) poly A tailing of the 3′end, 3) poly dT-primed double-strand cDNA synthesis with the incorporation of a T7 RNA polymerase promoter, and 4) RNA amplification by in vitro transcription.

[0095] Process C (central arrow in the diagram) starts with FPET RNA and involves 1) T7-(N)15 primed double-stranded cDNA synthesis with the incorporation of a T7 RNA polymerase promoter, and 2) RNA amplification by in vitro transcription.

[0096] Specifically, Protocol A involves a random primed (hexamers) cDNA synthesis that generates cDNA with a free 3′ OH on the terminal nucleotide of the FPET cDNA. The FPET cDNA is then tailed with Terminal Transferase (TdT) and dATP. The poly dA-tailed cDNA is then converted to double-stranded DNA with DNA polymerase I (Klenow) and T7-(dT)24 prim...

reference example 1

[0111] In Example 1 below, the following methods were used.

FPET RNA Extraction Procedure

[0112] RNA was extracted from 3-10 μm sections (for each patient). Paraffin was removed by xylene extraction followed by ethanol wash. RNA was isolated from sectioned tissue blocks using the MasterPure™ Purification kit (Epicentre Technologies, Madison, Wis.) and included a DNase I step. FPET RNA was further purified by filtration through a CHROMA SPIN™ DEPC-H20 30 column as described by suppliers (Clontech, Palo Alto). Briefly, 30 μl of 50-300 ng / μl FPET RNA was loaded onto a column (pre-spun at 2500 rpm (664×g) for 5 min. in a 5417 C eppendorf centrifuge), spun through the column (same conditions as the pre-spin) and stored at −80° C. FIG. 2 shows an example of RNA isolated from formalin fixed, paraffin embedded (FPE) breast cancer samples that were archived from 1 to 17 years.

Positive Control Complementary RNA (cRNA) Synthesis

[0113] Small RNA fragments complementary to amplicons for the ...

example 1

Standard Protocol

[0124] RNA was treated with polynucleotide kinase (PNK) or calf intestinal alkaline phosphatase (CIP), enzymes with 2′-3′ cyclic phosphatase activity and 3′ phosphatase activity, respectively. Capillary electrophoretic [Agilent 2100] analysis of the treated FPET RNA suggested that treatment of the FPET RNA with PNK or CIP removed the blocking phosphates, as judged by a subtle decrease in the mobility of the enzyme-treated RNA relative to that of the untreated RNA (FIG. 6A). Decreased electrophoretic mobility was expected because removal of the charged phosphate group would have decreased the charge / mass ratio of the FPET RNA.

[0125] If the blocking phosphates from the 3′ end of the FPET RNAs were effectively removed, then polyadenylation of the RNA should be possible. Treatment of FPET RNA with PNK followed by EPAP treatment (+PNK / +EPAP) resulted in a significant decrease in electrophoretic mobility of the FPET RNA (FIG. 6B). To confirm that the mobility shift was...

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Abstract

The invention relates to methods of using fragmented RNA, such as RNA obtained from archived fixed paraffin-embedded tissue material (FPET RNA) or other clinically biopsied tissue specimens for universal gene expression profiling.

Description

[0001] This application claims priority under 35 U.S.C. § 119(e) to provisional application Ser. No. 60 / 532,684 filed on Dec. 23, 2003, the entire disclosure of which is hereby expressly incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to methods of preparing RNA for gene expression profiling by a variety of methods. The methods of the invention are particularly useful for universal amplification of RNA, including RNA in which one or more RNA species is fragmented and / or blocked at it 3′ terminus, such as is obtained from fixed paraffin-embedded tissue (FPET). The methods are also useful for detecting RNA species which lack polyadenylation. In addition, methods of enhanced RT-PCR for useful in gene expression profiling are provided. DESCRIPTION OF THE RELATED ART [0003] Gene expression profiling is increasingly important both in biological research and in clinical practice. Gene expression profiling has been used to classify various cancer types ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12P19/34C12Q1/68
CPCC12N15/1096C12Q1/6813
Inventor KIEFER, MICHAELHOYT, KENNETH
Owner GENOMIC HEALTH INC
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