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

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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/