RNA sequencing of the nephron transcriptome: a technical note.
10.1016/j.krcp.2015.08.008
- Author:
Jae Wook LEE
1
Author Information
1. Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA. jwleemd@gmail.com
- Publication Type:Brief Communication
- Keywords:
Microdissection;
Nephron;
RNA sequencing;
Transcriptome
- MeSH:
Animals;
Base Sequence;
Collagenases;
DNA, Complementary;
Gene Expression;
Gene Library;
Genome;
Kidney;
Microdissection;
Nephrons*;
Rats;
Reverse Transcription;
RNA*;
RNA, Messenger;
Sequence Analysis, RNA*;
Sonication;
Transcriptome*
- From:Kidney Research and Clinical Practice
2015;34(4):219-227
- CountryRepublic of Korea
- Language:English
-
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.
