Real time RT-PCR as a tool for quantitative Hevea gene expression studies

Azlina Bahari, . and Chow K. S., . Real time RT-PCR as a tool for quantitative Hevea gene expression studies. pp. 51-64. ISSN 1511-1768

We describe a real time quantitative RT-PCR (qRT-PCR) approach to measure gene expression of selected latex genes with SYBR Green I as the reporter dye. In this study the target genes were the small rubber particle protein (SRPP) and farnesyl diphosphate synthas (FPPS) while the reference was the 18S ribosomal RNA (rRNA) gene. As a first requirement amplification efficiencies of primer pairs specific to target and reference genes were determined by two-step RT-PCR. In the RT (reverse transcription) step single-stranded cDNA was synthesised from latex total RNA. Then ten-fold serial dilutions were amplified with gene-specific primers to generate cycle threshold (Ct) values for constructing a cDNA standard curve for each gene. Subsequently one-step qRT-PCR reactions were set up to generate Ct values for the target and reference genes from the same RNA sample. A relative quantification method which employs an efficiency-compensated comparative Ct approach was used to calculate target gene transcript level relative to the 18S rRNA gene.

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Abstract

We describe a real time quantitative RT-PCR (qRT-PCR) approach to measure gene expression of selected latex genes with SYBR Green I as the reporter dye. In this study the target genes were the small rubber particle protein (SRPP) and farnesyl diphosphate synthas (FPPS) while the reference was the 18S ribosomal RNA (rRNA) gene. As a first requirement amplification efficiencies of primer pairs specific to target and reference genes were determined by two-step RT-PCR. In the RT (reverse transcription) step single-stranded cDNA was synthesised from latex total RNA. Then ten-fold serial dilutions were amplified with gene-specific primers to generate cycle threshold (Ct) values for constructing a cDNA standard curve for each gene. Subsequently one-step qRT-PCR reactions were set up to generate Ct values for the target and reference genes from the same RNA sample. A relative quantification method which employs an efficiency-compensated comparative Ct approach was used to calculate target gene transcript level relative to the 18S rRNA gene.

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Item Type:	Article
AGROVOC Term:	Hevea brasiliensis
AGROVOC Term:	Latex
AGROVOC Term:	Gene expression
AGROVOC Term:	Transcription
AGROVOC Term:	DNA sequence
AGROVOC Term:	Polymerase chain reaction
AGROVOC Term:	Natural rubber
Geographical Term:	Malaysia
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	28 Apr 2025 02:31
URI:	http://webagris.upm.edu.my/id/eprint/23009

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