Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

A Rapid Method for High Quality Rna Isolation from Jute : Corchorus Capsularis L. and Corchorus Olitorius L.

Farhan Khan
Department of Molecular, Cell and Developmental Biology, School of Biological Sciences, The University of Texas at Austin, Austin, Texas-78712, U.S.A.

Ahmad Islam
Department of Molecular, Cell and Developmental Biology, School of Biological Sciences, The University of Texas at Austin,Austin, Texas 78712, U.S.A.

Kanagasabapathi Sathasivan
Department of Molecular, Cell and Developmental Biology, School of Biological Sciences, The University of Texas at Austin,Austin, Texas 78712, U.S.A.

Abstract/ Executive Summary:

The isolation of RNA from herbaceous plants is difficult when there is an abundance of polysaccharides in the plant material. Corchorus capsularis and C. olitorius are two jute species, where conventional isolation procedures gave poor results. The present paper describes a modified method, which yields a greater quantity of RNA compared to the use of conventional protocols in both jute species. This procedure yielded 500 - 600 μg of RNA per gram of fresh tissue and took only 3 h to complete. The RNA obtained was of high quality and proved suitable to Reverse Transcription-PCR.

Keywords: Jute, Corchorus capsularis, C. olitorius, RNA isolation, Polysaccharides, RT-PCR

Location: University of Dhaka, Bangladesh

Start Date:

End Date:

Program Area: Resource Development and Management

Commodity: Jute products

Objectives:

i) To extract of RNA from jute to be used in RT-PCR and the construction of a cDNA library and

ii) To develop a suitable RNA isolation.

Methodology:

Jute seeds were obtained from the University of Dhaka, Bangladesh and subjected to no prior treatment. The seeds were soaked in distilled water for 2 h, then placed on a moist filter paper in a Petri dish at 30 seeds/per plate. The seeds were germinated and grown for seven days at 28⁰C. The seed coats shed at the end of the seven-day period. The amount of tissue per Petri dish was approximately 0.6 g. Diethylpyrocarbonate (DEPC) treated water was prepared by adding 0.5 ml of DEPC to 1 liter of water and stirred for two hours. Monophasic Lysis Solution (MLS) was made by mixing 190 ml phenol (38 %), 59 g guanidine thiocyanate (0.8 M), 38 g ammonium thiocyanate (0.4 M), 16.7 ml of 3 M sodium acetate pH 5 (0.1 M), 25 ml glycerol (5 %) and the volume brought to 500 ml using DEPC treated water. Sodium chloride sodium citrate (SSC) was prepared by adding 0.8 M sodium citrate + 1.5 M NaCl (SSC) in DEPC-water. In addition chloroform without isoamyl alcohol, absolute isopropanol, 75% EtOH (in DEPC water) and 3 M sodium acetate pH 5.5 were used. The samples were mixed well by inversion, vortexed and incubated at room temperature for 10 min. They were then centrifuged at 12,000 x g for 10 min. The supernatant was transferred to a fresh centrifuge tube by pouring and 0.2 ml of chloroform was added to each tube and vortexed vigorously for 1 min. The tubes were left at room temperature for 2 - 3 min. After centrifuging again at 12,000 x g for 10 min, using a sterile transfer pipette, the aqueous phase was transferred to a fresh centrifuge tube. SSC (0.8 M Nacitrate/1.5 M NaCl) and isopropanol were added at half volume of aqueous phase each. The RNA was size fractionated on a standard 1.2 % formaldehyde agarose gel and visualized using UV illumination. Reverse Transcription-PCR was carried out using the Promega Access RT-PCR System in a single tube reaction. The reverse transcription was carried out at 48⁰C for 45 min, followed by heat inactivating the AMVRT at 94⁰C for 2 min. The PCR reaction was carried out for 32 cycles. The conditions for each cycle were: denaturation at 94⁰C for 30 sec, annealing at 42⁰C for 1 min and extension at 68⁰C for 2 min.

Source of Information: Plant Tissue Cult. 14 (1) : 63-68, 2004 (June)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It was observed that the younger the plant, the lower the polysaccharide content. However, even the youngest plants harvested had very high polysaccharides which made nucleic acid isolation difficult. The optimal growing method found was allowing the seeds to germinate on moist filter paper in Petri dishes at a controlled temperature of 28⁰C for seven days. This method was beneficial in overcoming polysaccharide as well as soil contamination by forcing the plants to only use the material stored in the endosperm for growth and preventing them from synthesizing and storing excessive polysaccharides and metabolites from external nutrients. The RNA yield was about 600 μg per gram of tissue. The RNA quality was easily verified by visualization on an ethidium bromide agarose gel, which showed no degradation. The intactness of the RNA was indicated by the integrity of the ribosomal bands. The RNA obtained was of high quality enabling us to use it in RT-PCR with various primers, paving the way to clone genes for important agronomic characters. Thus, compared to conventional method, the present procedure was very effective in isolating RNA of the desired quality from the two polysaccharide rich jute species, C. capsularis and C. olitorius. This procedure yielded 500 - 600 μg of RNA per gram of fresh tissue in both the species. The method is also rapid (~ 3 h to complete), simple, efficient, and it does not require long ultracentrifugation. Observing its high efficacy, the protocol is recommended for isolation of RNA from plant species belonging to other taxa that are rich in polysaccharides.

Knowledge Management: Journal