Quantitation of Small RNAs in Caenorhabditis elegans Using Real-Time RT-PCR

Date of Award

2008

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

Real-Time PCR, RNA, PTGS, RT-PCR, Nematodes, Worms, C. elegans, Small RNAs

Area of Concentration

Biology

Abstract

RNAi is a gene-regulatory process mediated by small RNAs approximately 22 nucleotides in length and is conserved across a wide variety of organisms, including worms, flies, and humans. Three proteins, rha-1, rde-2, and mut-7, are involved in RNAi and silencing of repetitive transgene arrays in the germline, and mutant strains show defects in these processes. In order to examine the possibility that small RNAs are abnormally expressed in these worms and in double mutant strains, a modified real-time RT-PCR procedure was employed to quantitate the expression of three distinct classes of small RNA molecules: microRNAs, tiny non-coding RNAs, and X cluster RNAs. Results show that the rha-1(tm329) strain overexpresses all RNAs tested, while the rde-2(ne221) and mut-7(pk204) strains have reduced expression in the tncRNAs and X cluster RNAs, but not miRNAs. Further, the introduction of the rha-1(tm329) mutation into the rde-2 and mut-7 strains rescues expression to near wild-type levels of many of the small RNAs in the rde-2;rha-1 and mut-7;rha-1 double mutant strains. The mechanism for this rescue is currently unknown.

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