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Method to Quantify siRNAs, miRNAs and Polymorphic miRNAs

a technology of mirnas and sirnas, applied in the field of methods to quantify sirnas, mirnas and polymorphic mirnas, can solve the problem of the silencing effect being a temporary phenomenon

Inactive Publication Date: 2007-05-17
APPL BIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] Approaches to quantify miRNAs and other short target polynucleotides have been described in U.S. Non-Provisional application Ser. Nos. 10 / 947,460, and 11 / 142,720 to Chen et al.

Problems solved by technology

The primary advantage in the use of synthetic siRNAs is the ability to control the amount of siRNA, which may minimize the possibility of nonspecific effects, but the key disadvantage of synthetic siRNA is the transient nature of the silencing effect.

Method used

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  • Method to Quantify siRNAs, miRNAs and Polymorphic miRNAs
  • Method to Quantify siRNAs, miRNAs and Polymorphic miRNAs
  • Method to Quantify siRNAs, miRNAs and Polymorphic miRNAs

Examples

Experimental program
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example

[0049]

TABLE 11. Ligation / kinaseVolumeStockReaction(uL)ItemsRNA sample 3×1×(12ng / ul)(4ng / ul)2.50reverse stem-loop ligation150nm50nm2.50probeLigation Reaction MixT4 polynucleotide kinase10U / uL0.5U / uL0.375(10 U / uL, NEB, M0201L)T4 DNA ligase (2000 U / uL,2000U / uL50U / uL0.188NEB, M0202M)T4 DNA Ligase buffer (NEB,10×1×0.750B02025)AB RNase Inhibitor (P / N:20U / uL0.5U / uL0.188N8080119)H2O1.000Total7.50

Incubate at 16° C. / 30 min, 37° C. / 60 min, 4° C. on hold.

[0050]

TABLE 22. DigestionstockvolumeItems(u / ul)MixtureReaction(ul)UNG (NEB, M0280L)20.40.10.1AB RNase Inhibitor200.50.125(P / N: N8080119)T4 DNA Ligase buffer1010.25(NEB, B02025)H202.025Total2.50

Add 2.5 ul of UNG mix to 7.5 ul of ligation mix, 37° C. / 60 min, 4° C. on hold.

[0051]

TABLE 33. RTVolumeStock[1×](uL)ItemsRT primer5um1um3.00Ligation-digestion Product 3×1×5.00RT Enzyme MixdNTPs with dTTP (P / N:25mM0.25mM0.1508080261)eacheachMultiScribe Reverse50×3.33×  1.000Transcriptase (P / N: 4319983)10× RT Buffer (P / N: 4319981)10×1×1.500AB RNase Inhibit...

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Abstract

The present teachings provide methods, compositions, and kits for quantifying target polynucleotides. In some embodiments, a reverse stem-loop ligation probe is ligated to the 3′ end of a target polynucleotide, using a ligase that can ligate the 3′ end of RNA to the 5′ end of DNA using a DNA template, such as T4 DNA ligase. Following digestion to form an elongated target polynucleotide with a liberated end, a reverse transcription reaction can be performed, followed by a PCR. In some embodiments, the methods of the present teachings can discriminate between polymorphic polynucleoitides that vary by as little as one nucleotide.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims a priority benefit under 35 U.S.C. § 119(e) from U.S. Patent Application No. 60 / 711,480, filed Aug. 24, 2005, U.S. Patent Application No. 60 / 750,302, filed Dec. 13, 2005 and U.S. Patent Application No. 60 / 783,311, filed Mar. 16, 2006, which are incorporated herein by reference.FIELD [0002] The present teachings relate to methods, compositions, and kits for amplifying, identifying, and quantitying polymorphic target polynucleotides. INTRODUCTION [0003] In 1998, Andrew Fire and Craig Mello discovered that injection of double-stranded RNA (dsRNA) into Caenorhabditis elegans could initiate a potent sequence-specific degradation of cytoplasmic mRNAs (Fire, et al., 1998, Nature, 391, 806-811). In mammalian cells, RNA interference (RNAi) can be triggered by a variety of dsRNA or dsRNA-domain-containing molecules that are processed by the endoribonuclease Drosha and Dicer. Dicer is also responsible for the processing of ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6855C12Q1/6851C12Q2525/301C12Q2525/207C12Q2521/501C12Q2537/143
Inventor TAN, RUOYINGCHEN, CAIFUGUEGLER, KARL J.
Owner APPL BIOSYSTEMS INC
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