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Pmbr Paper Gurpreet Singh

Pmbr Paper Gurpreet Singh

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Publish by Abstract 
A Quick Method to Isolate RNA From Wheat andOther Carbohydrate-Rich Seeds
GURPREET SINGH
1
, SANJAY KUMAR
2,*
and PRABHJEET SINGH
1
1
 Department of Biotechnology, Guru Nanak Dev University, Amritsar 143005 (Punjab), India;
2
 Biotechnology Division Institute of Himalayan Bioresource Technology, PO # 6,Palampur 176061 (HP), India
Isolating RNA from carbohydrate-rich seeds Singhet al.
Abstract.
No reports on isolating RNA from carbohydrate-rich wheat seeds have beenpublished. Because of the presence of carbohydrates, published protocols yield smallamounts of poor quality RNA. Extracting seeds in a buffer (pH 9, 150 mM NaCl,1% sarcosyl) ensured maximum RNA solubility and the removal of most interferingsubstances. Extracted RNA was purified using a guanidine hydrochloride–based buffersystem. This protocol yields up to 148
µ
g of RNA from 100 mg of tissue in 3.5 h. AnA
260
 /A
280
ratio of 1.85 indicates RNA purity. Isolated RNA was amenable to downstreamapplications such as differential display. The developed method was extended to othercarbohydrate-rich seeds, such as barley and maize, with success.
Full text
:
This manuscript, in detail, is available only in the electronic version of the
Plant Molecular Biology Reporter.
Key words:
carbohydrates, differential display,
Hordeum vulgare
, polysaccharides, RNAisolation, sarcosyl,
Triticum aestivum, Zea mays
Abbreviations:
BME,
β
-mercaptoethanol; DTT, dithiothreitol; DEPC, diethylpyrocarbonate;EB, extraction buffer; EDTA, ethylenediaminetetraacetic acid; EtBr, ethidium bromide;H
2
O
2
, hydrogen peroxide; NaCl, sodium chloride; PCI, phenol-chloroform-isoamylalcohol.
Introduction
Wheat (
Triticum aestivum
L.) is the most economically important grain. Wheatdevelopment is adversely affected by water stress, which results in a substantialloss in yield. Studying the stress-induced changes in gene expression in wheatgrains of stress-tolerant and stress-susceptible cultivars is important to understandthe molecular basis of stress adaptation in wheat. Efficient extraction of highquality RNA from wheat seeds is crucial in gene expression studies and cDNAlibrary construction. However, large amounts of carbohydrates make RNA extrac-tion difficult. Methods for isolating RNA from plant tissues rich in starch have
Plant Molecular Biology Reporter 
21:
93a–93f, March 2003© 2003
International Society for Plant Molecular Biology. Printed in Canada.
*
Author for correspondence. e-mail: skplp@rediffmail.com; fax: +(91) 1834 230433;ph: +(91) 1834 233339.
Editor’s note: Although the scientific content of this manuscript has been reviewed, thefull text Web document has not been edited in detail.
 
been reported (Chomczynski and Sacchi, 1987; Logemann et al., 1987; Gehrig etal., 2000; Salzman et al., 1999; Hosein, 2001), but these methods are inefficientfor isolating good quality RNA from wheat.Here we report a quick and simplified method for isolating RNA fromwheat. Seeds were extracted in a buffer containing 150 mM NaCl and 1% sodiumlauryl sarcosinate (sarcosyl), which ensured maximum RNA solubility in theaqueous phase and the removal of most polysaccharides and other insoluble mate-rial. RNA in the aqueous phase was extracted using a guanidine hydrochlo-ride–based system (Logemann et al., 1987; Lal et al., 2001) with modifications.
Materials and Methods
Plant material
Wheat seeds (
T. aestivum
L., cultivar HD-2329) were sown in 5-L pots under nethouse conditions. Developing grains were collected at 15 d postanthesis,snap-frozen in liquid nitrogen, and stored at -80ºC until use.Seeds from barley (
 Hordeum vulgare
L. cv. Local Kaza) and maize (
 Zeamays
L. cv. Parvati) were purchased from the CSK Himachal Pradesh KrishiVishwavidyala, Palampur, India. Seeds were soaked overnight in 25°C sterilewater and ground in liquid nitrogen.
Solutions and reagents
Chilled absolute ethanol and 70% ethanol
Chloroform
DEPC-treated (0.1%) water
DEPC-treated (0.1%) autoclaved water
3 M sodium acetate (pH 5.2)
Extraction buffer (EB): 50 mM Tris-HCl (pH 9), 150 mM NaCl, 1% sarcosyl,20 mM EDTA, and 5 mM DTT
Guanidine hydrochloride buffer (GB): 8 M guanidine hydrochloride, 20 mMEDTA, 20 mM MES (pH 7), and BME (200 mM final concentration), whichwas added to the buffer just before starting the experiment
2 M NaCl
Sarcosyl (10%)
Phenol-chloroform-isoamylalcohol (25:24:1)Plasticware and pipette tips were treated overnight with 0.1% DEPC-treateddistilled water and autoclaved. Mortars and pestles were baked overnight at250ºC. Electrophoresis apparatus was dipped in 3% H
2
O
2
for 10 h and washedwith DEPC-treated autoclaved water. All solutions were prepared inDEPC-treated water and autoclaved thereafter, except Tris-HCl, which was pre-pared in 0.1% DEPC-treated autoclaved water and autoclaved.
 Isolating RNA from wheat seeds
Grind the seed sample in liquid nitrogen using a mortar and pestle.
Transfer 100 mg of the fine powder to a 2-mL microcentrifuge tube.
93b
Singh et al.
 
Add 500
µ
L of extraction buffer, vortex vigorously, and mix with 500
µ
L of PCI.
Vortex and centrifuge at 21,910
g
at 4ºC for 5 min.
Carefully remove upper aqueous phase (approximately 500
µ
L), add 650
µ
L of guanidine buffer and 350
µ
L of PCI, and vortex.
Centrifuge at 21,910
g
at 4°C for 5 min.
Remove upper aqueous phase (avoiding the interface) and add 500
µ
L of chloroform.
Vortex and centrifuge at 21,910
g
at 4°C for 5 min.
Transfer upper aqueous phase into 2 microcentrifuge tubes (approximately450
µ
L in each).
Add 45
µ
L of 3 M sodium acetate (pH 5.2) and 900
µ
L of chilled absoluteethanol to each tube.
Incubate at -80°C for 90 min.
Spin the pellet at 21,910
g
at 4°C for 20 min.
Wash the pellet with 70% chilled ethanol (v/v). Spin at 10,130
g
at 4°C for3 min.
Discard the supernatant and dry the pellet at room temperature.
Dissolve RNA pellet in minimum amount of autoclaved DEPC water. Store at-80°C until further use.
Quantify the RNA by monitoring the absorbance at 260 nm. Calculate theA
260
 /A
280
ratio.
Run 3-5
µ
g of RNA on a 1.2% denaturing agarose gel containing formaldehydeto check the integrity (Sambrook et al., 1989).
 Downstream application: mRNA differential display using wheat seed RNA
Isolated RNA was used for differential display reactions, performed according toLiang et al. (1994) and Lal et al. (2001). Total RNA isolated from developinggrains was digested with DNase 1. After removing DNase 1, RNA was reversetranscribed using T
11
C primer with a
Hin
d III site at the 5’ end. The reverse tran-scribed product was used for radioactive PCR using T
11
C and an arbitrary primer(5’-AAGCTTTCATATG-3’) in the presence of 
α
-[
33
P]dATP (3000 Ci/mmol).PCR products were fractionated on a 6% denaturing polyacrylamide gel. The gelwas dried, exposed to x-ray film, and developed after 72 h of exposure (Figure 2).Reagents were purchased from GenHunter Corporation (USA), BARC (India, forradiolabeled dATP), and Amersham Pharmacia Biotech (USA). Manufacturer’sinstructions were followed.
Taq
polymerase was purchased from Qiagen(Germany).
Results and Discussion
Procedures described by Gehrig et al. (2000) and Logemann et al. (1987) did notproduce satisfactory results in terms of yield and A
260
 /A
280
ratio (Figure 1A, lanes1 and 2, respectively). However, when RNA was isolated by our procedure,148
µ
g of RNA was obtained from 100 mg of seed tissue. An A
260
 /A
280
of 1.85indicates pure RNA. Distinct bands of 28S and 18S rRNA showed the RNA to beintact (Figure 1A, lane 3). The protocol took only 3.5 h.
 Isolating RNA from carbohydrate-rich seeds
93c

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