全 文 ：第 40 卷 第 5 期
2015 年 10 月
昆明理工大学学报(自然科学版)
Journal of Kunming University of Science and Technology(Natural Science Edition)
Vol． 40 No. 5
Oct． 2015
Ｒeceived:2015 － 01 － 12． Foundation projects:Talent Cultivation Project of Yunnan Province(KKSY201226103) ;Applica-
tion and Basic Ｒesearch Fund of Yunnan Province(KKS0201326002) ;Science and Technology Innovation Platform
Fund of Yunnan Province(2012DA002)．
Author biographical notes:Qiao Pu(1989 －) ，Female，Post － Graduate． Ｒesearch Area:Molecular biology and biochemistry．
E －mail:qiaopu@ hotmail. com
Author for correspondence:Han Qin-qin(1983 － ) ，Female，Instructor． Ｒesearch Area:Adversity Stress Mechanism of
Plant． E －mail:15398550161@ 163. com
doi:10． 16112 / j． cnki． 53 － 1223 /n． 2015． 05． 015
Efficient Isolation of ＲNA from Seeds of Tomato
and Chili Pepper
QIAO Pu，SONG Yu-zhu，ZHANG Jin-yang，CHEN Qiang，HAN Qin-qin
(Faculty of Life Science and Technology，Kunming University of Science and Technology，Kunming 650500，China)
Abstract:ＲNA is an important object of molecular biology in scientific research． Extraction of high quality ＲNA
in molecular biological method is a common user． High contents of protein，polyphenol and polysaccharide as well
as the characteristics of easy oxidation make it a challenge for ＲNA extraction from seeds of Solanaceae plant，
such as tomato and chili pepper． To solve these problems，a modified ＲNA extraction method is constructed based
on saracosyl /guanidine hydrochloride method． In this report，the OD260 /OD230 ratio of extracted ＲNA is higher
than 2． 0． Furthermore，the OD260 /OD280 ratios range from 1. 93 － 2. 00． This indicates that the ＲNA is of high
purity and without polyphenol，polysaccharide or protein contamination． The extracted ＲNA could be used for a
number of downstream analyses，including Northern blot hybridization，ＲT － PCＲ and so on． This method pro-
vides a method for isolation of high － quality and high － quantity ＲNA from seeds of Solanaceae plant． It is also a
simple and low － cost extraction protocol．
Key words:Solanaceae plant;tomato;chili pepper;ＲNA extraction;seed
CLC Number:Q781 Document Code:A Article ID:1007 － 855X(2015)05 － 0088 － 05
一种高效提取茄科类种子 ＲNA的方法
乔 璞，宋玉竹，张金阳，陈 强，韩芹芹
(昆明理工大学 生命科学与技术学院，云南 昆明 650500)
摘要:分子生物学是科学研究中最重要的科学领域，而 ＲNA 在分子生物学中又扮演着最重要的
角色．因此，高效快速的 ＲNA 提取方法在分子生物学中成为一种关键技术． 然而，茄科类植物
(如番茄，辣椒)其种子中含有大量的蛋白质、多酚和多糖，并且易氧化，这些都严重影响 ＲNA提
取的质量．为了解决这些问题，本文以十二烷基肌氨酸钠 /盐酸胍法为基础进行一定的改良，抽提
的 ＲNA的 OD260 /OD230均高于 2． 0，OD260 /OD280在 1． 93 ～ 2． 00 之间，这表明 ＲNA纯度很高，并且
没有多酚多糖和蛋白质的污染．使用此方法抽提的 ＲNA可应用于一系列的包括 Northern 印迹杂
交和 ＲT － PCＲ等的生物检测技术中．通过此方法能够从茄科植物的种子中提取出高产量、高质
量的 ＲNA，操作简单，成本较低．
关键词:茄科植物;番茄;辣椒;提取 ＲNA;种子
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0 Introduction
Seventy percent of food directly comes from seed of plant． Seed is essential for plant reproduction and propaga-
tion，which is the main carrier of plant genetic resources［1］． It is necessary for isolation of ＲNA with high purity
and quality in molecular biology research，such as ＲT － PCＲ，cDNA synthesis，library construction，gene cloning
and northern blot analysis of seed from plant［2，3］． Thus，obtaining high quality and quantity ＲNA is necessary．
Due to amount of plants and most of secondary metabolites，it is very difficult to isolate ＲNA from most
plant samples． High contents of polysaccharides and phenolic commonly are found in the plant tissues，which are
difficult to remove［4］． In the last study，it showed to be efficient in extracting ＲNA，according to adhesion of
plant samples to frozen section machine with synthetic glue for sectioning［5，6］． However，the seeds of solanaceae
plant are very small． Also，compared with other tissues，there are much higher contents of protein，polyphenol
and polysaccharide in seed． While，several methods for ＲNA isolation were applied to different plant seeds，such
as Fiber post method［7］，SDS method［8］，CTAB method［9］，Saracosyl /guanidine hydrochloride method［10］，high
salt law method［11］，Urea /LiCl method［12］ and so on． However，such methods failed to yield acceptable ＲNA
from seed mainly because of their costs and cumbersome protocol［13］． In addition，much polysaccharide or low
ＲNA contents in seed［14，15］． It is hard to get high quality ＲNA［16 － 18］． Thus，we improved extract components
based on Saracosyl /guanidine hydrochloride method to overcome these problems． This method contains a simple
and rapid step for removal of the high contents of polysaccharides and phenolic． Besides，it is suitable for most
fresh or dried seeds of solanaceae plant allowing efficient recovery of ＲNA．
1 Materials and methods
1. 1 Plant materials
Tomato fruit(Solanum lycopersicum)and Chili pepper(Capsicum annuum L．)were collected from mature
trees growing in the Chenggong orchard of Kunming University of Science and Technology(Kunming，China)．
Plants were transported on ice to the laboratory immediately after harvest and screened for uniform size and lack of
visible defects or decay． At each sampling time，3 fruits were sampled． The seeds(fresh seed and dry seed)of So-
lanaceae plant were diced under sterile conditions and frozen in liquid nitrogen． Then，stored at －80℃ until use．
1. 2 ＲNA isolation protocol
1. 2. 1 Solutions and reagents
All the solutions in ＲNA isolation were immersed in 0． 1% diethyl pyrocarbonate(DEPC)－ treated for 24 h
and then auto － claved at 121℃ for 30 min，then dried at 70℃ for subsequent test except for Tris － HCl and β －
mercaptoethanol，which were prepared with DEPC － treated water．
·Extraction buffer － 1% Saracosyl，100 mM Tris － HCl，280 mM NaCl，10 mM EDTA，10 μL /mL β － mer-
captoethanol(added just before use) ，pH 8． 5．
·Phenol
·Chloroform － isoamylalcohol(Chl:Iaa) (24∶1［volume fraction］)
·Phenol － chloroform(1∶1［volume fraction］)
·Isopropyl alcohol
·75%(volume fraction)ethyl alcohol
·DEPC － treated water
·10%(volume fraction)sodium hypochlorite
1. 2. 2 Protocol
ＲNA isolation protocol developed herein was in the following．
98第 5 期 乔璞，宋玉竹，张金阳，等:一种高效提取茄科类种子 ＲNA的方法

·Sampling of seeds of tomato fruit and Chili pepper(fresh seed and dry seed)were collected and placed
into ＲNase － free beaker，and immersed in 10% sodium hypochlorite about 15 min，then cut the seeds and dis-
card the endosperm．
·Grind each kind of seed in liquid nitrogen by using a baked mortar and pestle． Collect powder(up to 100mg)
in a microfuge tubes and immediately add 100 μL extraction buffer to each tube． Add 100 μL phenol，vortex enough to
make sure that all clumps are dispersed． Incubate at 0℃ for 10 min and centrifuge at 10 000 g for 5 min．
·Transfer the supernatant directly into a new microfuge tube and add 0． 5 volume of Chl:Iaa(24∶ 1)and
immediately vortex． Centrifuge at 10 000g for 5 min at 4℃ ．
·Transfer supernatant to a clean 1． 5 mL microfuge tube． Carefully pipette aqueous phase and do not collect
the white phase． Ｒe － extract with a 0． 5 volume of Phenol － chloroform(1∶1)． Centrifuge at 10 000 g for 5 min at
4℃ ．
·Transfer the supernatant to a new tube． Add equal volume of pre － cooling isopropyl alcohol and mix well
by pipetting up and down． Incubate at room temperature for 5 min． Centrifuge at 10 000 g for 5 min at 4℃ ． Dis-
card supernatant by pipetting．
·Add equal volume of 75% ethyl alcohol． Centrifuge at 10 000 g for 5 min at 4℃ ． Discard flow － through
and air － dry it for 10 min．
·Dissolve ＲNA in 50 μL DEPC － treated water．
·Store ＲNA at － 80℃ ．
The purified ＲNA was quantified with a spectrophotometer at wavelengths of 230，260 and 280 nm． The ratio of
OD260 /OD280，and OD260 /OD230 were calculated． The integrity of ＲNA was electrophoresed on a 1． 0% agarose gel．
1. 3 ＲT － PCＲ analysis
Ｒeverse transcription － polymerase chain reaction(ＲT － PCＲ)amplification was performed to demonstrate
the excellent use of ＲNA for subsequent analyses． Primers were constructed on the basis of the conserved se-
quence for a tomato actin gene． Primers synthesized were as follows:
Actin － forward(5 － CATGCCAACCATCACACCAGT －3) ;
Actin － reverse(5 － CACAGCGATGATGCTCCAAGGGCTGTG －3)．
5 μg of total ＲNA extracted from four kinds of seeds(fresh seeds of tomato，fresh seeds of chili pepper，dry
seeds of tomato，dry seeds of chili peper)were used for the first strand cDNA synthesis using Ｒevert AidTM First
Strand cDNA Synthesis Kit(MBI，USA)on the basis of the manufacturers instructions，respectively． ＲT － PCＲ
was performed with a 20 μL reaction mixture involving 1μL of cDNA template，0． 2 μL of Taq polymerase(5 U
per μL，MBI，USA) ，2 μL of dNTPs(10 mmol /L of each) ，2μL of 10 × PCＲ buffer，2 μL of forward primer(10
μmol /L)and 2 μL of reverse primer(10 μm/L)． The PCＲ amplification conditions were 94℃ for 2 min，32 cy-
cles of 94℃ for 45 s，58℃ for 45 s，72℃ for 1． 5 min，and then a final extension at 72℃ for 5 min． Aliquots of
5 μL PCＲ product was electrophoresed on 1． 0% agarose gel for image scanning．
1. 4 Northern blot analysis
30 μg of ＲNA extracted from seeds of tomato fruit and chili pepper was electrophoresed on a 1． 0% agarose
gel containing formaldehyde，followed by transferring to nylon membranes(Sangon Biotech，Shanghai)with 20 ×
SSC． The blots were baked for 2 h at 80℃ ．
Make the nylon membranes with 5 × SSC into hybridization tube and add pre － hybridization solution(5 ×
SSC，5 × Denhardts，1% SDS and 50% deionized formamide，50 μg /mL modified ssDNA)． The blots were
baked for 6 － 8 h at 42℃ ． Then add degenerative probe to hybridization tube and put them into mini － hybridizer
overnight． Discard hybridization solution and wash them with 2 × SSC，0． 1 % SDS at room temperature for 5 min．
Transfer nylon membranes and wash them with 0． 2 × SSC，0． 1 % SDS at 42℃ for 15 min on the bed temperature
09 昆明理工大学学报(自然科学版) 第 40 卷
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incubator．
2 Ｒesults and discussion
2. 1 UV scanning analysis of ＲNA purity and concentration
Tab． 1 ＲNA quality using spectrophotometric determinations
Sample OD260 /OD230 OD260 /OD280
Fresh seeds of tomato 2. 13 2. 00
Fresh seeds of chili pepper 2. 17 1. 97
Dry seeds of tomato 2. 05 1. 95
Dry seed of chili pepper 2. 26 1. 93
The extracted ＲNA from different solanaceae
plant by self － made method with UV spectrophotom-
etry were determined 3． 78，4． 52，3． 25 and 2． 98
μg /μL， respectively． In addition， their OD260 /
OD280 were 2． 00，1． 97，1． 95 and 1． 93 correspond-
ingly;OD260 /OD230 were 2． 13，2． 17，2． 05 and
2. 26，respectively(Table 1)． For all samples，the
OD260 /OD230 ratio was higher than 2． 0(2． 05 － 2． 26) ，indicating low levels of polyphenol and polysaccharide
(Table 1)． The OD260 /OD280 ratios ranged from 1． 93 － 2． 00． This indicated that the ＲNA was of high purity and
without protein contamination(Table 1)．
2. 2 ＲNA integrity
The ＲNA integrity was assessed by the sharpness
of ribosomal ＲNA bands visualized on a 1． 0% agar-
ose gel． For all ＲNA samples tested，distinct 28S and
18S rＲNA bands without degradation were observed
(Fig． 1)． According to the result，each extracted seed
ＲNA by self － made method showed three distinct
bands，suggesting that ＲNA was not degraded or con-
taminated． Meanwhile，the purity of ＲNA was much
higher．
2. 3 ＲT － PCＲ and Northern blot analysis
The quality of the ＲNA was further proved by
means of ＲT － PCＲ(Fig． 2a)and Northern blot analysis(Fig． 2b)． PCＲ products were analyzed using 1． 0%
agarose gel electrophoresis． The result indicated that clear bands were amplified with a length of about 750 bp
(Fig． 2a) ，which was consistent with the expected length． At the same time，northern blot analysis consistently
matched the result of ＲT － PCＲ，they all showed that total ＲNA extracted herein had nearly no DNA contamina-
tion and could be used directly for cDNA synthesis without mＲNA purification step or DNase treatment．
19第 5 期 乔璞，宋玉竹，张金阳，等:一种高效提取茄科类种子 ＲNA的方法

3 Conclusion
In this study，a method based on Saracosyl /guanidine hydrochloride method was constructed to obtain the
high contents of protein，polyphenol and polysaccharide from seeds of solanaceae plant． It was showed to be ad-
vantageous for isolating high quality of ＲNA from the seeds of solanaceae plant． The extracted ＲNA could be used
for further analyses as demonstrated by Northern blot and ＲT － PCＲ．
To sum up，the total ＲNA extracted by modified Saracosyl /guanidine hydrochloride method could meet the
requirement of the following molecular biology research，such as extraction of mＲNA，gene cloning，library con-
struction of cDNA from solanaceae plant and so on． Besides that，modified Saracosyl /guanidine hydrochloride
method was a cost － saving，effective and simple method to extract high － quality ＲNA from seed of solanaceae
plant．
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29 昆明理工大学学报(自然科学版) 第 40 卷
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