Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Stabilizing compositions and methods for extraction of ribonucleic acid

Inactive Publication Date: 2010-04-22
DNA GENOTEK
View PDF27 Cites 45 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]An object of the present invention is to provide a composition and method for prolonged storage of RNA from bodily fluids and / or tissues at room temperature, which compositions and methods further facilitate extraction of the RNA in as high a yield and as nearly intact state as is possible.

Problems solved by technology

Detection and / or analysis of such RNA are potentially of great importance, yet an accepted method that is optimal for collecting, preserving / stabilizing, transporting and extracting RNA has not yet been developed.
RNA is a labile compound and the widespread adoption for routine use of RNA as an analyte in detection and analysis of RNA has been limited because of its labile nature.
It is also sensitive to breakdown by acidic solutions.
Ribonuclease activity has previously been identified in human saliva (Bardon and Shugar, 1980), but the biochemical properties of this enzyme have not been well characterized.
At least in part as result of its instability RNA is often considered as an unsuitable analyte for diagnosis or detection.
The requirement for rapid transportation and / or the requirement of freezing may be problematic in terms of cost and storage space.
Additionally, in the case of remote locations and / or large-scale sample collection, rapid transportation and / or freezing may not be feasible.
Importantly, rapid processing / testing of clinical samples may not be feasible during an epidemic; back-logged samples will likely degrade over time and / or under sub-optimal storage conditions.
However, once collected, the time that useable RNA can be recovered may be limited because of the presence of ribonucleases in most tissues and bodily fluids.
However, cationic compounds, including tetradecyltrimethylammonium oxalate, have been found to be unsatisfactory in terms of ease of use and long term stability of RNA.
It has been found that once the cationic detergent is bound to nucleic acids, the nucleic acids are difficult to dissolve.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Stabilizing compositions and methods for extraction of ribonucleic acid
  • Stabilizing compositions and methods for extraction of ribonucleic acid
  • Stabilizing compositions and methods for extraction of ribonucleic acid

Examples

Experimental program
Comparison scheme
Effect test

example 1

Protocol for Obtaining Saliva Samples from Human Subjects

[0108]The subject is instructed to wait for a period of 30-60 minutes before last eating. If possible, the subject will brush his teeth (without using toothpaste). If possible, the subject will rinse his / her mouth with 50 ml of water. The subject will be requested to wait for 5-10 minutes to allow the mouth to clear of water. For subjects able to spit, they will be instructed to spit saliva into the special collection tube until the level of saliva reaches the 1 or 2 ml mark. Waiting after last eating and rinsing the mouth is desirable but not essential. Collection of saliva may take several minutes. If the subject finds that he / she is unable to deliver sufficient saliva, he / she will be given a few grains of table sugar to chew or place on the top of their tongue, and told not to be concerned if some of the sugar is spit into the tube. For subjects unable to spit (e.g., infants, young children, individuals with limitations / dis...

example 2

Comparison of the Present Composition to Oragene™

Sample Collection

[0111]In this example, a single subject provided two sputum / saliva samples (2 ml each) within a short period of time. One sample was collected into a vial containing 2 ml of a composition of the present invention comprising: 4% SDS, 50 mM CDTA, pH 6.6. Shortly afterwards, the same subject provided a second sample into a vial containing 2 ml of Oragene™ solution. Samples were shaken and left at room temperature (RT) for 3 days before purification of nucleic acids.

Methods

[0112]Nucleic acids were purified from this subject's saliva in Oragene™ or in the composition of the present invention. A portion of the subject's sample in Oragene™ was mixed with proteinase K and heated at 50° C. for 2 hours, without (FIGS. 1 and 2) and with (FIG. 2) an additional, subsequent heating at 90° C. for 15 minutes. A portion of the subject's sample in the composition was mixed with proteinase K, heated at 50° C. for 2 hours and then at 90°...

example 3

Optimizing pH Range for Maintaining the Stability of Pure RNA Derived from Sputum / Saliva

Methods

[0118]Pure RNA was diluted 6-fold into solutions buffered over a wide range of pH values (pH 3.0, 5.0, 6.0, 7.0, 8.2 and 10.0): Following a 16 hour period of incubation at 50° C., to allow partial hydrolysis of the phosphodiester backbone of the RNA to occur, an aliquot of the RNA buffered to each pH value (noted below) was analyzed by electrophoresis on a 0.9% agarose gel and stained with ethidium bromide (FIG. 3).

[0119]The sample order of FIG. 3 is as follows:

LaneSample1Lambda DNAHindIII digest2RNA marker3RNA, pH 3.04RNA, pH 5.05RNA, pH 6.06RNA, pH 7.07RNA, pH 8.28RNA, pH 10.0

Conclusions

[0120]RNA is stable chemically at neutral to slightly acid pH, as indicated by the preservation of the stained double bands characteristic of ribosomal RNA seen in treatments at pH 5.0-8.2. Note that a decrease in intensity of the upper band is expected before a decrease in the lower band. While not wishi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Temperatureaaaaaaaaaa
Temperatureaaaaaaaaaa
Fractionaaaaaaaaaa
Login to View More

Abstract

The present invention provides a composition and method for stabilizing ribonucleic acid (RNA) from biological samples such that the ribonucleic acid within the sample remains stable at room temperature. The composition comprises an anionic detergent and a buffering agent at a pH of about 5 to about 8.2 and is used in methods for extracting and storing ribonucleic acid from the biological sample.

Description

RELATED APPLICATIONS[0001]This application claims priority to U.S. Application Nos. 60 / 828,563; 60 / 866,985 and 60 / 949,778 the contents all of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The field of the invention generally relates to compositions and methods for storage and / or isolation of ribonucleic acids from bodily fluid(s), and / or secretion(s), (e.g., saliva, mucous), and / or tissue(s).BACKGROUND[0003]The importance of detection and analysis of ribonucleic acid (RNA) is becoming increasingly evident. For example, a large number of pathogenic mammalian viruses (e.g. SARS-CoA, Influenza virus, Measles virus, Rabies virus, Dengue fever virus, Respiratory Syncytial Virus (RSV), HIV and Hepatitis A, C-E virus) have genomes based on RNA rather than DNA. Detection and / or analysis of such RNA are potentially of great importance, yet an accepted method that is optimal for collecting, preserving / stabilizing, transporting and extracting RNA has ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12P19/34C09K3/00C07H21/02
CPCC12N15/1003C12Q1/6806C12Q2527/125Y02A50/30
Inventor BIRNBOIM, HYMAN CHAIMJACKSON, ADELE
Owner DNA GENOTEK
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com