Methods for cloning small RNA species

Inactive Publication Date: 2009-01-08
INTEGRATED DNA TECHNOLOGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]The invention provides improvements to the current methods of small RNA (such as miRNA) cloning to provide greater efficiency and simplification to redu

Problems solved by technology

However, the synthetic marker RNA does not contain a 5′-phosphate group which renders it inactive for the 5′-linkering step.
Each purification step is time

Method used

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  • Methods for cloning small RNA species
  • Methods for cloning small RNA species
  • Methods for cloning small RNA species

Examples

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example 1

[0049]This example provides a protocol for practicing the proposed microRNA cloning process. Total RNA is prepared using methods well-known in the art, such as the use of the mirVana RNA isolation kit (Ambion®) following the Ambion® protocol. RNA isolation methods utilizing glass fiber filters (GFF) or silicate adsorption should be avoided as these “rapid” methods for RNA isolation often deplete the small RNA pool present in a natural sample. Organic extraction reagents such as Trizol or RNA STAT 60 are preferred. After the RNA is extracted and purified, the sample is enriched for small RNA species. If practical, it is preferred to employ at least 50 μg to 100 μg of total RNAs for small RNA enrichment however lower input mass amounts can be used when sample is limited. The mass of RNAs in the miRNA size range of 18 nt to 26 nt is a very small fraction of the total RNA present, and size selection at this stage of library construction is essential to the quality of the end product. Th...

example 2

[0074]The following example demonstrates the use of the method of the invention to isolate novel miRNAs from tissue.

[0075]Total RNA was prepared from brain, heart, lung, liver and kidney tissue of the South American marsupial species Monodelphis domestica using the mirVana RNA isolation kit (Ambion®, Austin, Tex.) according to the manufacturer's protocol. Between 100 μg and 200 μg of total cellular RNA was obtained from each tissue fragment. A 100 μg RNA pool, comprising 20 μg of RNA from each tissue, was used as the RNA source for the experiment. This pooled RNA sample was spiked with 10 pmole of the 21 nt internal control ORN marker “miSPIKE” and size fractionated on a 12% denaturing (7M urea) polyacrylamide gel at 275 volts for 90 minutes.

SEQ ID NO. 15′-rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrU-3′

[0076]The gel was stained with GelStar® and RNAs were visualized under UV excitation. A 4 mm square gel slice was excised using the miSPIKE ORN marker as a size guide. The gel slice was...

example 3

[0089]The following example demonstrates the use of the method of the invention to isolate novel piRNAs from tissue.

[0090]Unlike miRNAs which are expressed in all tissues, other classes of small RNAs have limited tissue distribution. The Piwi associated RNAs (piRNAs) are a different class of small RNA which are specific to gonads (ovary and testis). The piRNAs are longer than miRNAs, and usually are 26-32 nt long. The method of the invention was used to isolate and sequence identify novel piRNAs. Total RNA was prepared from testis of the South American marsupial species Monodelphis domestica using the mirVana RNA isolation kit (Ambion®, Austin, Tex.) according to the manufacturer's protocol. Between 100 μg and 200 μg of total cellular RNA was obtained from each tissue fragment. 100 μg testis derived RNA was employed for the experiment. This RNA sample was spiked with 10 pmole of the 31 nt internal control ORN marker “piSPIKE” and size fractionated on a 12% denaturing (7M urea) polya...

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Abstract

This invention pertains to methods for cloning microRNA (miRNA) and other small ribonucleic acid (RNA) species from relevant cell sources.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]The present application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 60 / 946,922 filed 28 Jun. 2007. The entire teachings of the above application are incorporated herein by reference.FIELD OF THE INVENTION[0002]This invention pertains to methods for cloning microRNA (miRNA) and other small ribonucleic acid (RNA) species from relevant cell sources.BACKGROUND OF THE INVENTION[0003]Small, non-coding, regulatory RNA species such as microRNAs have emerged in recent years as a powerful agent in regulating gene expression in eukaryotic cells. First discovered in 1993 (Lee at al., Cell 75: 843-854 (1993)), microRNAs are an abundant new class of regulatory elements that have been shown to impact all aspects of normal cellular processes in both plants and animals, including cell death, differentiation, and proliferation, as well as abnormal processes including cancer (Bartel, Cell 116: 281-297 (2004); Du and Za...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H1/00
CPCC12Q1/6806C12Q2565/125C12Q2525/151
Inventor DEVOR, ERIC J.HUANG, LINGYANBEHLKE, MARK A.LAIKHTER, ANDREI
Owner INTEGRATED DNA TECHNOLOGIES
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