RNA sequencing of the nephron transcriptome: a technical note
Kidney Research and Clinical Practice
;
: 219-227, 2015.
Article
in English
| WPRIM
| ID: wpr-79189
ABSTRACT
To understand the functions of the kidney, the transcriptome of each part of the nephron needs to be profiled using a highly sensitive and unbiased tool. RNA sequencing (RNA-seq) has revolutionized transcriptomic research, enabling researchers to define transcription activity and functions of genomic elements with unprecedented sensitivity and precision. Recently, RNA-seq for polyadenylated messenger RNAs [poly(A)'-mRNAs] and classical microdissection were successfully combined to investigate the transcriptome of glomeruli and 14 different renal tubule segments. A rat kidney is perfused with and incubated in collagenase solution, and the digested kidney was manually dissected under a stereomicroscope. Individual glomeruli and renal tubule segments are identified by their anatomical and morphological characteristics and collected in phosphate-buffered saline. Poly(A)'-tailed mRNAs are released from cell lysate, captured by oligo-dT primers, and made into complementary DNAs (cDNAs) using a highly sensitive reverse transcription method. These cDNAs are sheared by sonication and prepared into adapter-ligated cDNA libraries for Illumina sequencing. Nucleotide sequences reported from the sequencing reaction are mapped to the rat reference genome for gene expression analysis. These RNA-seq transcriptomic data were highly consistent with prior knowledge of gene expression along the nephron. The gene expression data obtained in this work are available as a public Web page (https//helixweb.nih.gov/ESBL/Database/NephronRNAseq/) and can be used to explore the transcriptomic landscape of the nephron.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Sonication
/
RNA
/
RNA, Messenger
/
Base Sequence
/
Gene Expression
/
Gene Library
/
Genome
/
Sequence Analysis, RNA
/
Collagenases
/
DNA, Complementary
Type of study:
Prognostic study
Limits:
Animals
Language:
English
Journal:
Kidney Research and Clinical Practice
Year:
2015
Type:
Article
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