Reference Genes Selection for Quantitative Real-time PCR Using RankAggreg Method in Different Tissues of Capra Hircus

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2013 Dec 21

PMID 24358246

Citations 26

Authors

Mohammad Javad Najafpanah

Mostafa Sadeghi

Mohammad Reza Bakhtiarizadeh

Affiliations

Soon will be listed here.

Abstract

Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes.

Citing Articles

Evaluation of Reference Gene Stability in Goat Skeletal Muscle Satellite Cells during Proliferation and Differentiation Phases.

Zhan S, Zhang L, Zhong T, Wang L, Guo J, Cao J Animals (Basel). 2024; 14(17).

PMID: 39272264 PMC: 11394193. DOI: 10.3390/ani14172479.

Temporal Transcriptome Dynamics of Reveals the Mechanism of the Differences in Muscle Development and IMF Deposition between Fuqing Goats and Nubian Goats.

Liu Y, Wu X, Xu Q, Lan X, Li W Animals (Basel). 2024; 14(12).

PMID: 38929389 PMC: 11200590. DOI: 10.3390/ani14121770.

Anti-inflammation-based treatment of atherosclerosis using Gliclazide-loaded biomimetic nanoghosts.

Karami Z, Mehrzad J, Akrami M, Hosseinkhani S Sci Rep. 2023; 13(1):13880.

PMID: 37620556 PMC: 10449813. DOI: 10.1038/s41598-023-41136-y.

Selection and validation of reference genes for the normalization of quantitative real-time PCR in different muscle tissues of rabbits.

Ni M, Li Z, Li J, He H, Wang Y, Jiang Y BMC Zool. 2023; 7(1):60.

PMID: 37170359 PMC: 10127086. DOI: 10.1186/s40850-022-00159-0.

QuilA® adjuvanted Coxevac® sustains Th1-CD8-type immunity and increases protection in Coxiella burnetii-challenged goats.

Tomaiuolo S, Jansen W, Soares Martins S, Devriendt B, Cox E, Mori M NPJ Vaccines. 2023; 8(1):17.

PMID: 36788233 PMC: 9929268. DOI: 10.1038/s41541-023-00607-z.

References

Klie M, Debener T . Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida). BMC Res Notes. 2011; 4:518. PMC: 3248381. DOI: 10.1186/1756-0500-4-518. View

Cankorur-Cetinkaya A, Dereli E, Eraslan S, Karabekmez E, Dikicioglu D, Kirdar B . A novel strategy for selection and validation of reference genes in dynamic multidimensional experimental design in yeast. PLoS One. 2012; 7(6):e38351. PMC: 3366934. DOI: 10.1371/journal.pone.0038351. View

Condori J, Nopo-Olazabal C, Medrano G, Medina-Bolivar F . Selection of reference genes for qPCR in hairy root cultures of peanut. BMC Res Notes. 2011; 4:392. PMC: 3199266. DOI: 10.1186/1756-0500-4-392. View

Lardizabal M, Nocito A, Daniele S, Ornella L, Palatnik J, Veggi L . Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity. PLoS One. 2012; 7(5):e36323. PMC: 3341372. DOI: 10.1371/journal.pone.0036323. View

Ohl F, Jung M, Xu C, Stephan C, Rabien A, Burkhardt M . Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?. J Mol Med (Berl). 2005; 83(12):1014-24. DOI: 10.1007/s00109-005-0703-z. View

Uddin M, Cinar M, Tesfaye D, Looft C, Tholen E, Schellander K . Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues. BMC Res Notes. 2011; 4:441. PMC: 3219825. DOI: 10.1186/1756-0500-4-441. View

Lee J, Fitzgerald J, DiMicco M, Grodzinsky A . Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression. Arthritis Rheum. 2005; 52(8):2386-95. DOI: 10.1002/art.21215. View

Becker C, Riedmaier I, Reiter M, Tichopad A, Pfaffl M, Meyer H . Changes in the miRNA profile under the influence of anabolic steroids in bovine liver. Analyst. 2011; 136(6):1204-9. DOI: 10.1039/c0an00703j. View

Mehta D, Menke A, Binder E . Gene expression studies in major depression. Curr Psychiatry Rep. 2010; 12(2):135-44. PMC: 2847693. DOI: 10.1007/s11920-010-0100-3. View

10.

Fan C, Ma J, Guo Q, Li X, Wang H, Lu M . Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis). PLoS One. 2013; 8(2):e56573. PMC: 3577859. DOI: 10.1371/journal.pone.0056573. View

11.

Radonic A, Thulke S, Mackay I, Landt O, Siegert W, Nitsche A . Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004; 313(4):856-62. DOI: 10.1016/j.bbrc.2003.11.177. View

12.

Chomczynski P, Sacchi N . The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2007; 1(2):581-5. DOI: 10.1038/nprot.2006.83. View

13.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

14.

Ponton F, Chapuis M, Pernice M, Sword G, Simpson S . Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol. 2011; 57(6):840-50. DOI: 10.1016/j.jinsphys.2011.03.014. View

15.

Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen J . Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 2007; 35(Web Server issue):W71-4. PMC: 1933133. DOI: 10.1093/nar/gkm306. View

16.

Bonefeld B, Elfving B, Wegener G . Reference genes for normalization: a study of rat brain tissue. Synapse. 2008; 62(4):302-9. DOI: 10.1002/syn.20496. View

17.

Neville M, Collins J, Gloyn A, McCarthy M, Karpe F . Comprehensive human adipose tissue mRNA and microRNA endogenous control selection for quantitative real-time-PCR normalization. Obesity (Silver Spring). 2010; 19(4):888-92. PMC: 4623139. DOI: 10.1038/oby.2010.257. View

18.

Kadegowda A, Bionaz M, Thering B, Piperova L, Erdman R, Loor J . Identification of internal control genes for quantitative polymerase chain reaction in mammary tissue of lactating cows receiving lipid supplements. J Dairy Sci. 2009; 92(5):2007-19. DOI: 10.3168/jds.2008-1655. View

19.

Seol D, Choe H, Zheng H, Jang K, Ramakrishnan P, Lim T . Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc. BMC Res Notes. 2011; 4:162. PMC: 3118343. DOI: 10.1186/1756-0500-4-162. View

20.

Zhang X, Ding L, Sandford A . Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol Biol. 2005; 6:4. PMC: 551605. DOI: 10.1186/1471-2199-6-4. View