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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq
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Quantitative RNA Analysis Using RNA-Seq.

Peter J Myler1,2,3, Jacqueline A McDonald4, Pedro J Alcolea4,5

  • 1Center for Global Infectious Disease Research, Seattle Childrens Research Institute, 307 Westlake Ave N, Suite 500, Seattle, 98109-5219, WA, USA. peter.myler@seattlechildrens.org.

Methods in Molecular Biology (Clifton, N.J.)
|April 14, 2019
PubMed
Summary
This summary is machine-generated.

We present novel RNA-sequencing (RNA-seq) methods for accurately measuring mRNA levels in Leishmania. One approach utilizes the unique spliced leader (SL) sequence for precise transcript mapping and quantification.

Keywords:
Differential gene expressionRNA-seqTranscriptomemRNA

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Area of Science:

  • Molecular Biology
  • Genomics
  • Parasitology

Background:

  • High-throughput sequencing, specifically RNA-sequencing (RNA-seq), offers a digital and nucleotide-level analysis of mRNA.
  • Understanding mRNA expression is crucial for studying gene regulation and biological processes in various organisms.
  • Leishmania and other trypanosomatids possess a unique spliced leader (SL) sequence at the 5' end of their mRNAs.

Purpose of the Study:

  • To develop and describe novel RNA-sequencing (RNA-seq) methodologies tailored for Leishmania and related trypanosomatid parasites.
  • To leverage the conserved spliced leader (SL) sequence for enhanced transcript analysis.
  • To provide accurate digital readouts of mRNA levels and precise transcript mapping.

Main Methods:

  • Implementation of two distinct RNA-sequencing (RNA-seq) approaches.
  • Exploitation of the 39-nucleotide mini-exon or spliced leader (SL) sequence present in Leishmania mRNA.
  • High-throughput sequencing of complementary DNA (cDNA) derived from messenger RNA (mRNA).

Main Results:

  • RNA-seq provides a digital quantification of mRNA abundance across a wide dynamic range.
  • Transcript mapping to the nucleotide level is achievable with the described methods.
  • The SL sequence facilitates specific and efficient RNA-seq analysis in Leishmania.

Conclusions:

  • The developed RNA-seq methods enable accurate and detailed analysis of the transcriptome in Leishmania.
  • Utilizing the SL sequence offers a powerful strategy for studying gene expression in trypanosomatids.
  • These techniques advance the study of gene regulation and parasite biology.