An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone: Applications in mice with bone property altering Lrp5 mutations

Ugur M. Ayturk, Christina M. Jacobsen, Danos C. Christodoulou, Joshua Gorham, Jonathan G. Seidman, Christine E. Seidman, Alexander Robling, Matthew L. Warman

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5+/+), knockout (Lrp5-/-), and high bone mass (HBM)-causing (Lrp5p.A214V/+) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5 +/+ and Lrp5p.A214V/+) and "insufficient" (Lrp5-/-) diaphyseal bone, and far fewer differentially expressed genes between Lrp5p.A214V/+ and Lrp5+/+ diaphyseal bone.

Original languageEnglish
Pages (from-to)2081-2093
Number of pages13
JournalJournal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Volume28
Issue number10
DOIs
StatePublished - Oct 2013

Fingerprint

Low Density Lipoprotein Receptor-Related Protein-5
RNA
Bone and Bones
Messenger RNA
Mutation
Skeletal Muscle
Bone Marrow
Collagenases
Missense Mutation
Genotype
Animal Rights
Gene Expression
Tibia
Computer Simulation
Genes
Digestion
Alleles
Phenotype

Keywords

  • cells of bone
  • diseases and disorders of/related to bone
  • genetic animal models
  • molecular pathway development
  • statistical methods
  • WNT/β-catenin/ LRPS

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone : Applications in mice with bone property altering Lrp5 mutations. / Ayturk, Ugur M.; Jacobsen, Christina M.; Christodoulou, Danos C.; Gorham, Joshua; Seidman, Jonathan G.; Seidman, Christine E.; Robling, Alexander; Warman, Matthew L.

In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 28, No. 10, 10.2013, p. 2081-2093.

Research output: Contribution to journalArticle

Ayturk, Ugur M. ; Jacobsen, Christina M. ; Christodoulou, Danos C. ; Gorham, Joshua ; Seidman, Jonathan G. ; Seidman, Christine E. ; Robling, Alexander ; Warman, Matthew L. / An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone : Applications in mice with bone property altering Lrp5 mutations. In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2013 ; Vol. 28, No. 10. pp. 2081-2093.
@article{ebc0d0504dc6472d9f2fe8f0772df891,
title = "An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone: Applications in mice with bone property altering Lrp5 mutations",
abstract = "Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5+/+), knockout (Lrp5-/-), and high bone mass (HBM)-causing (Lrp5p.A214V/+) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 {"}sufficient{"} (ie, Lrp5 +/+ and Lrp5p.A214V/+) and {"}insufficient{"} (Lrp5-/-) diaphyseal bone, and far fewer differentially expressed genes between Lrp5p.A214V/+ and Lrp5+/+ diaphyseal bone.",
keywords = "cells of bone, diseases and disorders of/related to bone, genetic animal models, molecular pathway development, statistical methods, WNT/β-catenin/ LRPS",
author = "Ayturk, {Ugur M.} and Jacobsen, {Christina M.} and Christodoulou, {Danos C.} and Joshua Gorham and Seidman, {Jonathan G.} and Seidman, {Christine E.} and Alexander Robling and Warman, {Matthew L.}",
year = "2013",
month = "10",
doi = "10.1002/jbmr.1946",
language = "English",
volume = "28",
pages = "2081--2093",
journal = "Journal of Bone and Mineral Research",
issn = "0884-0431",
publisher = "Wiley-Blackwell",
number = "10",

}

TY - JOUR

T1 - An RNA-seq protocol to identify mRNA expression changes in mouse diaphyseal bone

T2 - Applications in mice with bone property altering Lrp5 mutations

AU - Ayturk, Ugur M.

AU - Jacobsen, Christina M.

AU - Christodoulou, Danos C.

AU - Gorham, Joshua

AU - Seidman, Jonathan G.

AU - Seidman, Christine E.

AU - Robling, Alexander

AU - Warman, Matthew L.

PY - 2013/10

Y1 - 2013/10

N2 - Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5+/+), knockout (Lrp5-/-), and high bone mass (HBM)-causing (Lrp5p.A214V/+) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5 +/+ and Lrp5p.A214V/+) and "insufficient" (Lrp5-/-) diaphyseal bone, and far fewer differentially expressed genes between Lrp5p.A214V/+ and Lrp5+/+ diaphyseal bone.

AB - Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5+/+), knockout (Lrp5-/-), and high bone mass (HBM)-causing (Lrp5p.A214V/+) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5 +/+ and Lrp5p.A214V/+) and "insufficient" (Lrp5-/-) diaphyseal bone, and far fewer differentially expressed genes between Lrp5p.A214V/+ and Lrp5+/+ diaphyseal bone.

KW - cells of bone

KW - diseases and disorders of/related to bone

KW - genetic animal models

KW - molecular pathway development

KW - statistical methods

KW - WNT/β-catenin/ LRPS

UR - http://www.scopus.com/inward/record.url?scp=84884514583&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84884514583&partnerID=8YFLogxK

U2 - 10.1002/jbmr.1946

DO - 10.1002/jbmr.1946

M3 - Article

C2 - 23553928

AN - SCOPUS:84884514583

VL - 28

SP - 2081

EP - 2093

JO - Journal of Bone and Mineral Research

JF - Journal of Bone and Mineral Research

SN - 0884-0431

IS - 10

ER -