Optimizing Housekeeping Gene Selection for qPCR

The primers of nine RGs (Table 2) were chosen due to their different biological functions and irrelevance. Metabolism-related genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M); ribosomal genes: ribosomal protein large P0 (RPLP0); cellular structure and cytoskeleton: beta-actin (ACTB); transcription and translation: eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), TATA box binding protein (TBP); cell signaling and regulation: peptidylprolyl isomerase A (PPIA), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ); nucleotide metabolism: hypoxanthine phosphoribosyltransferase 1 (HPRT1). GAPDH, B2M, EEF1A1, RPLP0, PPIA, and YWHAZ were referenced from a previous report [35 (link),38 (link)]. ACTB, TBP, and HPRT1 were designed using the PrimerQuest™ tool at an annealing temperature of 60 °C and analyzed using OligoAnalyzer 3.1, a software that confirms the absence of hairpins, homodimers, and heterodimers. To evaluate primer efficiency, RT-qPCR was performed with the 10-fold diluted cDNA of the SF-MSCs, following a previously established protocol [38 (link)]. Standard curve parameters were calculated using the RotorGene Q Series software 2.1.0 (Qiagen, Hilden, Germany).

Free full text: Click here

Bok E.Y., Kim S.B., Thakur G., Choe Y.H., Oh S.J., Hwang S.C., Ock S.A., Rho G.J., Lee S.I., Lee W.J, & Lee S.L. (2023). Insensitive Effects of Inflammatory Cytokines on the Reference Genes of Synovial Fluid Resident-Mesenchymal Stem Cells Derived from Rheumatoid Arthritis Patients. International Journal of Molecular Sciences, 24(20), 15159.

Publication 2023

RotorGene Q Series software 2.1.0

Actin Beta 2 microglobulin Beta actin Biological functions Cdna Cellular structure Cytoskeleton Elongation factor 1 alpha 1 Genes Glyceraldehyde 3 phosphate dehydrogenase Hypoxanthine phosphoribosyltransferase Metabolism Nucleotide Peptidylprolyl isomerase Primer Protein Ribosomal Ribosomal protein p0 Tata box binding protein Transcription Tryptophan Tyrosine 3 monooxygenase

Corresponding Organization : Kyungpook National University

Other organizations : Gyeongsang National University Hospital, Rural Development Administration, National Institute of Animal Science

Top 5 similar protocols

Variable analysis

independent variables

RT-qPCR
10-fold diluted cDNA of the SF-MSCs

dependent variables

Standard curve parameters

control variables

Annealing temperature of 60 °C
Absence of hairpins, homodimers, and heterodimers (confirmed using OligoAnalyzer 3.1)

controls

No positive or negative controls were explicitly mentioned.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!