Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative real-time PCR (qPCR)

Omar Abd Rasid, . and Nor Fakhrana Iskandar, . and Ahmad Parveez Ghulam Kadir, . and Ho C. L., . and Lim F. H., . and Noor Azmi Shaharuddin, . and Abu Seman Idris, . Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative real-time PCR (qPCR). pp. 170-181. ISSN 1511-2780

Quantitative real-time PCR (qPCR) has become a favourite method for quantification of mRNA transcripts. However several optimisation steps must be performed to avoid misleading qPCR results. One of the steps is selection of reference genes for normalisation purpose and these genes should be stably expressed across the samples. In this study isolation of partial-length cDNA encoding seven potential reference genes from Ganoderma boninense has been performed. These potential reference genes are -tubulin -tubulin -actin elongation factor 2 (eef2) glyceraldehyde 3-phosphate dehydrogenase (gapdh) 40S ribosomal (r40s) and ubiquitin C (ubc). The expression of these reference genes was studied in mycelia white button and fruiting body tissues of G. boninense. The qPCR data were analysed using BestKeeper and geNorm algorithms and both softwares have identified -tubulin eEF2 and -tubulin as the most stable reference genes and r40s and ubc as the least stable reference genes. Three reference genes with the lowest M value (eEF2 -tubulin and -tubulin) were recommended by the geNorm software to be used in the qPCR analysis for more accurate normalisation.

Full text available from: Official URL: http://jopr.mpob.gov.my/wp-content/uploads/2014/06...

Abstract

Quantitative real-time PCR (qPCR) has become a favourite method for quantification of mRNA transcripts. However several optimisation steps must be performed to avoid misleading qPCR results. One of the steps is selection of reference genes for normalisation purpose and these genes should be stably expressed across the samples. In this study isolation of partial-length cDNA encoding seven potential reference genes from Ganoderma boninense has been performed. These potential reference genes are -tubulin -tubulin -actin elongation factor 2 (eef2) glyceraldehyde 3-phosphate dehydrogenase (gapdh) 40S ribosomal (r40s) and ubiquitin C (ubc). The expression of these reference genes was studied in mycelia white button and fruiting body tissues of G. boninense. The qPCR data were analysed using BestKeeper and geNorm algorithms and both softwares have identified -tubulin eEF2 and -tubulin as the most stable reference genes and r40s and ubc as the least stable reference genes. Three reference genes with the lowest M value (eEF2 -tubulin and -tubulin) were recommended by the geNorm software to be used in the qPCR analysis for more accurate normalisation.

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Item Type:	Article
AGROVOC Term:	Ganoderma
AGROVOC Term:	Isolation
AGROVOC Term:	Selection
AGROVOC Term:	Gene expression
AGROVOC Term:	PCR
AGROVOC Term:	Polymerase chain reaction
AGROVOC Term:	Messenger rna
AGROVOC Term:	Gene transcription
AGROVOC Term:	Actin
AGROVOC Term:	Computer software
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	24 Apr 2025 06:29
URI:	http://webagris.upm.edu.my/id/eprint/24618

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