NDLI: Evaluation of Suitable Internal Control Genes for RT-qPCR in Yak Mammary Tissue during the Lactation Cycle

Content Provider	PubMed Central
Author	Jiang, Mingfeng Lee, Jung Nam Massimo, Bionaz Deng, Xiao Yu Wang, Yong
Editor	Seagroves, Tiffany
Copyright Year	2016
Abstract	The yak is primarily found throughout the Tibetan high plateau and the surrounding mountainous area of south central Asia; among its others attributes, its milk is very important for the local population. A key concern in the field of yak research is the better understanding of which genes control the production and composition of milk. The most accurate and sensitive method for gene expression analysis is quantitative reverse transcription polymerase chain reaction (RT-qPCR). It is essential for reliable RT-qPCR to be able to the normalize the data using internal control genes (ICGs). However, it is critical to assess the reliability of the normalization by testing multiple ICGs. Our objective was to uncover a reliable normalization for RT-qPCR data obtained from yak mammary tissue during the lactation cycle. We assessed the reliability of 10 ICGs (ACTB, EIF6, GAPDH, LRP10, MRPL39, MRPS15, MTG1, RPS8, RPS23, and UXT) using geNorm. The analysis revealed that all of the tested ICGs can be considered to be reliable, but the use of the 6 most stable ICGs should be applied to yield a reliable normalization factor (NF). We compared the results of 3 target genes (CSN1S1, ESR1, and MYC) normalized using 6, 3, or 1 of the best ICGs. We did not observe overall differences between the 3 normalization strategies with the exception of 1 time point in MYC. The use of only a single ICG is not recommended; thus, we concluded that the calculation of the NF using the 3 best ICGs, MRPS15, RPS23, and UXT, is a reliable normalization strategy for RT-qPCR data obtained from yak mammary tissue during pregnancy and lactation. A dilution effect of the ICGs due to a large increase in the mRNA of abundantly expressed genes in bovine and porcine mammary tissue during the lactation cycle was previously observed. To test for the presence of a dilution effect in our study, we evaluated the pattern of non-normalized RT-qPCR data of ICGs from pregnancy to lactation and compared them with the total RNA concentration, milk yield, and non-normalized RT-qPCR data of 3 target genes. With a few exceptions, the non- normalized RT-qPCR data for the tested ICGs was significantly increased by lactation and had a positive correlation with total RNA and the non-normalized RT-qPCR data of CSN1S1. These data clearly indicated the presence of a “concentration effect” of single mRNA that remains unexplained but needs to be accounted for during the normalization of RT-qPCR data. Based on our findings, we recommend that the NF of the MRPS15, RPS23, and UXT genes should be used in the normalization of RT-qPCR data obtained from mammary tissue of lactating yaks during pregnancy and lactation.
Related Links	http://dx.doi.org/10.1371/journal.pone.0147705
Starting Page	147705
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	1
Volume Number	11
Language	English
Publisher	Public Library of Science
Publisher Date	2016-01-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Selection of Suitable Reference Genes for RT-qPCR Analyses in Cyanobacteria

Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis

Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

Alterations of Glucose-Dependent Insulinotropic Polypeptide and Expression of Genes Involved in Mammary Gland and Adipose Tissue Lipid Metabolism during Pregnancy and Lactation

RT-qPCR Normalization Genes in the Red Alga Chondrus crispus

lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

A Pipeline to Determine RT-QPCR Control Genes for Evolutionary Studies: Application to Primate Gene Expression across Multiple Tissues

Old and New Stories: Revelations from Functional Analysis of the Bovine Mammary Transcriptome during the Lactation Cycle

Establishment of Mammary Gland Model In Vitro: Culture and Evaluation of a Yak Mammary Epithelial Cell Line

Evaluation of Suitable Internal Control Genes for RT-qPCR in Yak Mammary Tissue during the Lactation Cycle

Similar Documents

Selection of Suitable Reference Genes for RT-qPCR Analyses in Cyanobacteria

Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis

Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

Alterations of Glucose-Dependent Insulinotropic Polypeptide and Expression of Genes Involved in Mammary Gland and Adipose Tissue Lipid Metabolism during Pregnancy and Lactation

RT-qPCR Normalization Genes in the Red Alga Chondrus crispus

lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

A Pipeline to Determine RT-QPCR Control Genes for Evolutionary Studies: Application to Primate Gene Expression across Multiple Tissues

Old and New Stories: Revelations from Functional Analysis of the Bovine Mammary Transcriptome during the Lactation Cycle

Establishment of Mammary Gland Model In Vitro: Culture and Evaluation of a Yak Mammary Epithelial Cell Line

Evaluation of Suitable Internal Control Genes for RT-qPCR in Yak Mammary Tissue during the Lactation Cycle