CSIR Central

26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species

IR@IHBT: CSIR-Institute of Himalayan Bioresource Technology, Palampur


Field	Value

Title	26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species

Creator	Singh, K Raizada, J Bhardwaj , P Ghawana, S Rani, A Singh, H Kaul, Kiran Kumar, Sanjay

Subject	Nutraceuticals

Description	Gene expression analysis is important to understand complex regulatory mechanism and identiWcation of genes relevant to biological processes. Quantitative expression data provide insight into the switched-on and switched-oV genes as well as into the up-regulated and down-regulated genes. This is achieved by a number of available methods, among which reverse transcriptionpolymerase chain reaction (RT-PCR)1-based analysis and real-time RT-PCR-based analysis have the advantage of automation, high speed, high sensitivity, high throughput, and lower requirement of RNA quantity. For relative quantitation, RNA levels of the gene under investigation are compared from sample to sample using an internal control to normalize for diVerences in sample concentration and loading. The use of such genes as internal controls relies on the premise that they exhibit a constant basal level of expression that is consistent, nonregulated, independent of the cell cycle, and nonresponsive to external treatments or developing stage. Housekeeping genes such as �-actin, rRNAs, glyceraldehydes- 3-phosphate dehydrogenase (GAPDH), tubulins, hypoxanthine phosphoribosyl transferase, L32, albumins, and cyclophilins are widely used as internal controls but need to be standardized for the target species [1–3]. Among the reported genes, those for rRNA are less likely to Xuctuate under the conditions that aVect the expression of mRNAs, probably due to much greater abundance of these genes (>80%) and the involvement of diVerent polymerases for transcription of mRNAs and rRNAs [4,5]. We developed primers from the conserved region of the 26S rRNA gene and evaluated them in seven diVerent plant species: Arabidopsis thaliana (arabidopsis), a model system in plant biology; Camellia sinensis (tea), a commercially important crop that possesses medicinal properties primarily attributed to polyphenols [6]; Arnebia euchroma (arnebia), whose roots are the source of alkanins/shikonins that are used as coloring agents and also exhibit antibacterial, antifungal, wound-healing, and antiinXammatory properties [7]; Caragana jubata (caragana), a leguminous high-altitude plant that tolerates extreme environmental conditions [8]; Rheum emodi (rheum), another higher altitude plant that contains a medicinal compound, anthraquinones [9]; Picrorhiza kurroa (picrorhiza), an alpine herb that is known for hepatoprotective and immunomodulatory activities [10]; and Stevia rebaudiana (stevia), which contains stevioside, a noncaloric sweetener that is more than 100 times sweeter than table sugar [11]. Because no internal control gene primers have been reported for these plants (except arabidopsis, which have tremendous scope of metabolic engineering), proactive action has been taken to develop an internal control for gene expression studies. Experiments were conducted on diVerent plant parts, and various treatments were provided to all seven plants (as described in the Wgure legends). RNA was isolated

Date	2004

Type	Article PeerReviewed

Format	application/pdf

Identifier	http://ihbt.csircentral.net/405/1/150_2005_26s_rRna.pdf Singh, K and Raizada, J and Bhardwaj , P and Ghawana, S and Rani, A and Singh, H and Kaul, Kiran and Kumar, Sanjay (2004) 26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species. Analytical Biochemistry, 335. pp. 330-333.

Relation	http://ihbt.csircentral.net/405/