Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia.

Jaime Marcial-Quino,Edgar Sierra-Palacios,Rosa Castillo-Rodríguez,Roberto Arreguin-Espinosa,Eduardo Rodríguez-Bustamante,Adriana Castillo-Villanueva,Francisco Fierro,Yadira Rufino-González,Saúl Gómez-Manzo,Horacio Reyes-Vivas,America Vanoye-Carlo

doi:10.3390/genes7120131

Abstract

Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.

Highlights

IntroductionGiardia lamblia is a flagellate protozoan that adapted over time to a parasitic lifestyle [1]
Giardia lamblia is a flagellate protozoan that adapted over time to a parasitic lifestyle [1].This organism causes the disease known as giardiasis in humans, but can affect several vertebrate organisms [2]
We describe the use of the stem-loop reverse transcription (RT)-qPCR technique to detect different small

Summary

Introduction

Giardia lamblia is a flagellate protozoan that adapted over time to a parasitic lifestyle [1] This organism causes the disease known as giardiasis in humans, but can affect several vertebrate organisms [2]. Small nucleolar RNA (snoRNA) has been identified as a source of microRNAs (miRNAs) in some protozoans and metazoans [5,6,7,8,9,10] These miRNAs are small RNA molecules that regulate gene expression in lower and higher eukaryotes, but they have not been studied in detail in Giardia [11], the origins and evolution of miRNAs and other regulatory small RNAs have been extensively characterized [12]. Some miRNAs (miR1, miR2, miR3, miR6, miR10) derived from snoRNA have been identified in G. lamblia by small RNA isolation and ligation to RNA linkers [6], and by sequencing analysis followed by primer extension [7]

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Conclusion