RESEARCH
Rapid and Effective Method of RNA Isolation from Green
Microalga Ankistrodesmus convolutus
Tran Thanh Æ Hishamuddin Omar Æ
Mohd Puad Abdullah Æ Vu Thi Quynh Chi Æ
Mostafa Noroozi Æ Huynh Ky Æ Suhaimi Napis
Published online: 9 June 2009
Ó Humana Press 2009
Abstract The rapid and effective method for the isolation
of RNA from green microalga Ankistrodesmus convolutus
based on homogenization in a simple CTAB buffer and
selective precipitation of RNA with lithium chloride is
developed. This procedure avoids the use of toxic chao-
tropic agents and phenol while high concentration of
dithiothreitol is used to inhibit RNase activity and prevent
oxidative cross-linking of nucleic acids by phenolics. The
extraction procedure was able to produce high quality and
intact RNA from A. convolutus. The yield of total RNA
was 0.69–0.73 mg/g of fresh weight, with A
260
/A
280
ratio
of 1.79–1.86. The obtained RNA was of sufficient quality
and suitable for downstream application such as RT-PCR
and cDNA library construction. The procedure may also
have wider applicability for total RNA isolation from other
green microalgae species.
Keywords Ankistrodesmus convolutus Á cDNA library Á
Green microalga Á RNA isolation Á RT-PCR Á Northern blot
Abbreviations
CTAB Cetyltrimethylammonium bromide
DEPC Diethyl pyrocarbonate
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
PVP Polyvinylpyrrolidone
SDS Sodium dodecyl sulfate
Introduction
The purity and integrity of isolated RNA is a critical
determinant of its effectiveness in molecular biological
procedure. Isolation of intact, high quality and quantity
RNA from green microalga suitable for the studies of pro-
moter and other controlling elements with Northern analy-
sis, RT-PCR, and cDNA library construction is difficult
because of the high levels of phenolics, lipids, polysac-
charides, and endogenous RNases. The tissues with high
level of polysaccharides yield poor quality RNA or no RNA
at all as polysaccharides co-precipitate with RNA in low
ionic strength buffers [1]. Indeed, algae as well as green
microalgae are typically rich in lipids and polysaccharides,
which make it difficult to get clean separation of RNA and
the rest of the cellular debris. As a result of this, algae
present themselves as unpromising candidates for molecu-
lar biology studies. Several RNA extraction procedures
have been developed to overcome this problem in plant as
well as similar tissues, but few procedures deal with green
microalgae. Some of the RNA extraction protocols for
plants and algae rich in polysaccharides include modifica-
tion of the acid guanidinium thiocyanate:phenol:chloroform
method [2], the use of soluble polyvinylpyrrolidone (PVP)
and ethanol precipitation [3], the use of high molecular
T. Thanh Á M. P. Abdullah Á V. T. Q. Chi Á H. Ky Á
S. Napis (&)
Department of Cell and Molecular Biology, Faculty
of Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, UPM Serdang, Selangor 43400, Malaysia
e-mail: suhaimi@putra.upm.edu.my; s.napis@gmail.com
H. Omar Á M. Noroozi
Department of Biology, Faculty of Sciences,
Universiti Putra Malaysia, UPM Serdang,
Selangor 43400, Malaysia
T. Thanh Á V. T. Q. Chi
Rubber Research Institute of Vietnam,
301 Nguyen Van Troi Street, Tan Binh District,
Ho Chi Minh City, Vietnam
Mol Biotechnol (2009) 43:148–153
DOI 10.1007/s12033-009-9182-8