research article insects rna profiling reveals absence of...

Research ArticleInsectsrsquo RNA Profiling Reveals Absence of lsquolsquoHidden Breakrsquorsquo in28S Ribosomal RNA Molecule of Onion Thrips Thrips tabaci

Rosaline Wanjiru Macharia Fidelis Levi Ombura and Erick Onyango Aroko

International Center for Insect Physiology and Ecology (icipe) PO Box 30772 Nairobi 00100 Kenya

Correspondence should be addressed to Rosaline Wanjiru Macharia rwanjiruicipeorg

Received 28 August 2014 Revised 4 November 2014 Accepted 19 January 2015

Academic Editor Ben Berkhout

Copyright copy 2015 Rosaline Wanjiru Macharia et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

With an exception of aphids insectsrsquo 28S rRNA is thought to harbor a ldquohidden breakrdquo which cleaves under denaturing conditionsto comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels The degraded appearance confoundsdetermination of RNA integrity in laboratories that rely on gel electrophoresis To provide guidelines for RNA profiles RNAfrom five major insect orders namely Diptera Hemiptera Thysanoptera Hymenoptera and Lepidoptera was compared underdenaturing and nondenaturing conditionsThis study confirmed that although present in most of insectrsquos RNA the ldquohidden breakrdquois absent in the 28S rRNA of onion thripsThrips tabaci On the other hand presence of ldquohidden breakrdquo was depicted in whitefliesrsquo28S rRNAdespite their evolutionary grouping under same order with aphids Divergence of 28S rRNA sequences confirms variationof both size and composition of gap region among insect species However phylogeny reconstruction does not support speciationas a possible source of the hidden break in insectrsquos 28S rRNA In conclusion we show that RNA from a given insect order doesnot conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discoveredspecies

1 Introduction

Insects (Arthropoda Insecta) constitute up to 55 of charac-terized species playing different roles in the environment [1]For instance fruit flies thrips stem borers and aphids arecrop pests tsetse flies and mosquitoes transmit pathogenswhile honey bees have direct economic benefits Molecularcharacterization of insects involving RNA-Seq and quantita-tive RT-PCR employRNA [1]However despitemany years ofresearch on insect rRNAmisinterpretation of their RNApro-files is common tomolecular biologists partly due to the vari-ations in 28S rRNA thermolability in different insect species

Success of downstream RNA application depends on thequality and integrity of the extracted RNA Isolation of highquality RNA from biological samples is however challengedby the ubiquitous presence of ribonucleases (RNases) thatrapidly degrade freshly prepared RNA [2] This makes theassessment of RNA quality mandatory prior to its down-stream application The assessment usually relies heavily on

the resolution of 18S and 28S rRNA bands on a gel eitherthrough automated bioanalyzers [3] or denaturing agarosegel electrophoresis [4] The bioanalyzer approach is moresensitive but relatively costly relegating most resource poorlaboratories to the denaturing gel electrophoresis approachIt is therefore important to know how to accurately interpretRNA profiles in agarose gels Under denaturing conditions28S rRNA from insects and most protostomes is charac-terized by its dissociation into two equally sized 120572 and 120573subunits [5] which comigrates with the 18S rRNA to exhibita single band profileThis dissociation of 28S rRNAmoleculeis attributed to the presence of an UAAU tract in their rRNAloopwhich acts as the cleavage site also known as the ldquohiddenbreakrdquo [6]TheUAAU tract is located about 10 bases upstreamof 51015840 end of 28S120573 segment of 28S rRNAmolecule In additionthe presence of ldquohidden breakrdquo has also been reported in the58S rRNA of Drosophila and in higher plant chloroplasts [7]In contrast the pea aphid 28S rRNA just like deuterostomes28S rRNA does not harbor the ldquohidden breakrdquo but instead

Hindawi Publishing CorporationJournal of Nucleic AcidsVolume 2015 Article ID 965294 8 pageshttpdxdoiorg1011552015965294

2 Journal of Nucleic Acids

contains a GC-rich hairpin loop that lacks the UAAU tract[5 7]

Efforts to understand molecular mechanisms involvedin the introduction of the gap have been hindered byunavailability of 28S rRNA secondary structures and highconservation of sequences reported in the regions that flankthe ldquohidden breakrdquo This makes it difficult to compare theldquohidden breaksrdquo [7] Nevertheless an earlier study suggestedthat the hidden break is probably introduced into the polynu-cleotide chain during or after the processing of 28S rRNAfrom its precursor [5]

In the present study banding profiles of RNA fromfive major insect orders considered of economic importanceincluding Diptera Hemiptera Thysanoptera Hymenopteraand Lepidoptera were compared with an aim of providingguidelines to the appearance of RNA for insects belonging tothese orders Importantly the absence of a hidden break wasobserved in the onion thripsThrips tabaci

2 Materials and Methods

21 Insect Sampling Representative insects for the fiveorders were obtained from insect colonies maintained atthe icipe insectaries and screen houses They included 150aphids (Hemiptera) Brevicoryne brassicae 60 mosquitoes(Diptera) Anopheles arabiensis 20 tsetse flies (Diptera)Glossina fuscipes fuscipes 16 honey bees (Hymenoptera)Apismellifera 200 whiteflies (Hemiptera) of Aleyrodidae familysix stem borers (Lepidoptera)Chilo partellus four silkworms(Lepidoptera) Bombyx mori 40 wasps (Hymenoptera)Fopius arisanus 10 fruit flies (Diptera) Dacus bivittatus and1000 thrips (Thysanoptera)Thrips tabaciThe sample sizewasinformed by the size of the fly and experiments were done inreplicates

22 RNA Extraction RNA from the insect samples wasextracted using the Direct-zol RNA Mini Prep Kit (ZymoResearch Corporation Irvin CA USA) following themanu-facturerrsquos instructions Fresh whole insect flies (le50mg) werehomogenized in 500 120583L of TRI-reagent and centrifuged inan Eppendorf AG 5417R centrifuge (Hamburg Germany) at12000timesg for 10min The supernatant was transferred intoa new tube followed by addition of one volume of absoluteethanol into the supernatant After vortexing 700 120583L of themixture was transferred into a Zymo-Spin IIC Column ina collection tube centrifuged at 12000timesg for 1min and theflow through discardedThe spin columnwas transferred intoa new collection tube and 400 120583L Direct-zol RNA prewashsolution was added and centrifuged at 12000timesg for 1minThe flow through was discarded and this step was repeatedInto the column 700 120583L RNA wash buffer was added andcentrifuged at 12000timesg for 1min The flow through wasdiscarded and the column spun again at 12000timesg for 2minto completely remove any residual wash buffer The columnwas then transferred into an RNase-free 15mL Eppendorftube and 25 120583L of DNaseRNase-free water was added ontothe column matrix centrifuged at maximum speed for 1min

for RNA elution The eluted RNA was immediately used fordownstream processes

23 Determination of RNAYield and Purity Thequantity andquality of the extracted RNA yield were determined using aNanodrop (ND) 2000c Spectrophotometer (Thermo Scien-tific Wilmington USA) by measuring optical density of 2120583Lof extracted RNA at A

260 nm and A280 nm respectively Any

sample with A260 nmA280 nm ratio below 17 was discarded

24 Denaturing Agarose Gel Electrophoresis A 12formaldehyde agarose (FA) gel was prepared by mixing12 g of agarose with 10mL of 10X FA gel buffer (200mMMOPS 50mM sodium acetate 10mM EDTA pH 70) Thiswas topped to 100mL using RNase-free water The mixturewas heated to melt the agarose and cooled to approximately65∘C after which 18mL of 37 123M formaldehyde and6 120583L of 10mgmL ethidium bromide were added Themixture was thoroughly but gently mixed then poured ontoa casting tray and left to polymerize under a laminar flowchemical hood

The RNA samples were mixed with 1 volume of 5XRNA loading dye prepared as per the Qiagen protocol [8]Sixteen 120583L of saturated aqueous bromophenol blue solutionwas mixed with 80120583L of 500mM EDTA of pH 80 720120583L of37 123M formaldehyde 2mL of 100 glycerol 3084mLformamide and 4mL of 10X FA gel buffer and topped up10mL with RNase-free water One volume of the dye wasadded to three volumes of RNA sample and mixed by pipet-ting The sample-dye mixture was down spun and incubatedat 65∘C for 5min then chilled on ice and immediately loadedonto the FA gel

The gel was run at 70V in the 1X FA gel runningbuffer (100mL of 10X FA gel buffer 20mL of 37 123Mformaldehyde and 880mL RNase-free water) for 1 hourfollowed by visualization of the gel under UV illuminationand analyzed using KODAK Gel Logic 200 Imaging System(Raytest GmbH Straubenhardt)

25 Nondenaturing Agarose Gel Electrophoresis A 12agarose gel was prepared by adding 12 g of agarose into100mL of 1X TAE buffer and heating to dissolve Six 120583L of10mgmL ethidium bromide was added to the mixture andthe sample RNA then mixed with a non-denaturing 6X DNAloading dye and the samples loaded onto the gel alongsidea molecular weight DNA marker (OrsquoGeneRuler 1 Kb plusDNA Ladder) Electrophoresis visualization and gel imageanalysis were carried out as outlined above

26 Multiple Sequence Alignment Phylogeny Reconstructionand Secondary Structure Determination Representative 28SrDNA sequences for the studied insect orders were obtainedfrom GenBank [9] (Table 2) In case of species lackingsequences from the database their sequences were substi-tuted with those of closely related species The 28S rDNAsequences of G fuscipes fuscipes and Brevicoryne brassicaewere substituted with G morsitans morsitans and A glycine

Journal of Nucleic Acids 3

Table 1 Nanodrop readings for RNA samples Brevicoryne bras-sicae Thrips tabaci whiteflies (Aleyrodidae) Anopheles arabiensisGlossina fuscipes fuscipes Dacus bivittatus Chilo partellus Bombyxmori Apis mellifera and Fopius arisanus

Species name Concentration (ng120583L) A260280 nm

Brevicoryne brassicae 17948 213Brevicoryne brassicae 18543 213Thrips tabaci 10861 216Thrips tabaci 10619 215Aleyrodidae 6471 206Aleyrodidae 6548 213Anopheles arabiensis 4780 203Anopheles arabiensis 4237 220Glossina fuscipes fuscipes 12486 203Glossina fuscipes fuscipes 15485 233Dacus bivittatus 4193 217Dacus bivittatus 4967 221Chilo partellus 9915 201Chilo partellus 7876 205Bombyx mori 11772 192Bombyx mori 10617 204Apis mellifera 8688 222Apis mellifera 8180 221Fopius arisanus 4489 209Fopius arisanus 3741 212

respectively No relative sequences were recovered to repre-sent Dacus bivittatus and Chilo partellus in phylogeny recon-struction Multiple sequence alignment was performed usingMuscle version 36 [10] and a phylogenetic tree reconstructedusing PhyML [11] General time reversible (GTR) model forsubstitution was applied with 100 bootstrap replicates Theresulting tree was rendered using fig tree [12]

A sequence containing mapped hidden break and itsflanking regions in B mori [7] was aligned and comparedagainst those of G morsitans morsitans T knoxi and Aglycine The species were chosen as representatives for pres-ence and absence of ldquohidden breakrdquo as observed in thedenatured gelT knoxi sequence was used in place ofT tabacibased on its length Secondary structures of these regionswere determined using ldquoThe Predict a Secondary Structureserverrdquo at httprnaurmcrochestereduRNAstructureWebServersPredict1Predict1html which combines four predic-tion algorithms Structures withmaximum free energies wereadopted for comparison and mapping of the hidden breakregions

3 Results

Purity and quantity of extracted RNAwere determined usinga Nanodrop Observed absorbance (260280) ratio for thesamples was above 20 (Table 1) which is the recommendedrange for pure RNA [8] Concentration of all samples wasgt350 ng120583L which was high enough to be viewed under gelelectrophoresis

Table 2 Species names of 28S rDNA sequences used in comparativeanalysis alongside associated GenBank IDs percentage GC contentand length in base pairs

Species name GenBanksequence ID

GCcontent

Length(bp)

An arabiensis AF4178121 54 527T knoxi KC5131191 55 1893T tabaci AY5233921 50 678F arisanus JN6405721 42 620Amellifera AJ3029361 56 2748Bmori AY0389911 52 1192Gmorsitans morsitans KC1778341 39 2165A glycine JQ259057 51 3547

RNA profiles were visualized by both denaturing andnon-denaturing agarose gel electrophoresis Apart fromaphids Brevicoryne brassicae and their close ldquocousinsrdquoThripstabaci other insectsrsquo RNA exhibited single banding profilesunder denaturing condition (Figure 1(a)) On the other handunder non-denaturing gel electrophoresis all RNA samplesappeared degraded (with 18S rRNA band showing higherintensity as compared to 28S rRNA band) (Figure 1(b))

31 Sequence Homology and Phylogeny Reconstruction of 28SrRNAs Sequence homology of the ldquohidden breakrdquo and itsflanking regions was assessed through multiple alignmentand visualized in Jalview [13] The rRNA alignment showshigh variability across the insect species with sequences of Amellifera G morsitans morsitans and B mori sharing morenucleotides compared to those of T knoxi and A glycine(Figure 2)

GC content of the region around the gap region wasassessed using EMBOSS geecee tool httpembossbioinfor-maticsnlcgi-binembossgeecee Like A glycine (49) Tknoxi variable region sequence recorded a higher GC compo-sition (47) compared to that of A mellifera (39) B mori(35) and G m morsitans (22) Evolutionary relatednessof 28S rRNAmolecules of the studied insects was assessed byconstructing a phylogenetic tree of their 28S rRNA sequencesor of their close relatives in case of missing sequences Theresulting tree with bootstrap values is shown in Figure 3

32 Model Secondary Structure of Thripsrsquo 28S rRNA VariableRegion Possible secondary structure of thripsrsquo gap regionand its flanking region was generated using T knoxi sequenceand compared against that of B moriG morsitans morsitansand A glycine (Figures 4(a)ndash4(d))

4 Discussion

Earlier studies on insect RNA have reported presence of aldquohidden breakrdquo that disintegrates under denaturing condi-tions in all insects except aphids [5ndash7 14] Molecular mech-anism behind introduction of this hidden break remainsunclear However its introduction has been attributed to


M 1 2 3 4 5 6 7 8 9 10 1112 M

M 13 14 15 16 17 18 19 20 M

28S18S

5S

28S18S

5S

(a)

M 1 2 3 4 5 6 7 8 9 10 11

12

M

M 13 14 15 16 17 18 19 M

28S18S

5S

28S18S

5S

(b)

Figure 1 Gel images showing the banding profiles of the various insect RNA sampled (a) Lane M OrsquoGeneRuler 1 kb DNA Plus (ThermoScientific) Lanes 1 and 2 aphids Lanes 3 and 4 thrips Lanes 5 and 6 whiteflies Lanes 7 and 8 mosquitoes Lanes 9 and 10 tsetse fliesLanes 11 and 12 fruit flies Lanes 13 and 14 stem borer Lanes 15 and 16 silk worm Lanes 17 and 18 wasps and Lanes 19 and 20 bees showsappearance of RNA on 12 formaldehyde agarose (FA) gel On the other hand (b) shows RNA appearance on 12 agarose non-denaturinggel Lanes 1 and 2 aphids Lane 3 whiteflies Lanes 4 and 5 mosquitoes Lanes 6 and 7 tsetse flies Lanes 8 and 9 fruit flies Lanes 10 and 11stem borer Lanes 12 and 13 silk worm Lanes 14 and 15 bees Lanes 16 and 17 wasps and Lanes 18 and 19 thrips

10 20 30 40 50 60 70 80 90 100 110

G morsitans1-72A glycine1-68B mori1-102

A mellifera1-118T knoxi1-68

--------------------AAGGCGA----AAGCU----GAAAAUUUUCAAGU-------------UAUAUAUAUAUAAAUAAUGUAUAAAACACUUGAAAAUAAUUGAACG-----AACAGCAGUUGGACAUGGGUUAGUCGAUCCUAAGCCAUAGGGAAGUUCCGUUUCAAA------GGCGCACUUUG--------------------------------------------AACAGUAGUUGCUCGCGAGUCAGUCGAUCCUAAGCUCAAGGAGAGAUCUUAUGUCGAUG---UGGCGUGUUUUUUUAUAAUU---UUUAAAAAUAAUGAAAAAUAACG----------AACAGCCGUUGCACACGAGUCAGUCGAUCCUAAGCCCUAAGAGAAAUCCUAUGUAAAUGAGGUGUCCUAAGAUCAUACACACAAACAUAUAAAAAUUGAAAUAUGAGACCAGAAACAC----------GUUCGCGAGUGAGUCGAUCCUAAGCCUAAAGAGAAAUCUUUAGUA-------UGGUCGAGCCUUGUAGGUUCCCA---------------------------------

ConsensusAACAGCAGUUGC+C+CGAGUCAGUCGAUCCUAAGCCC+A+GAGAAAUCUUAUGUAAAUG---UGGC+UAUUUUUAUAUAA++AAA-UUAAAAA-A-UGAAAAAUAA----A-------

Figure 2Multiple alignment of gap region ofBmori 28S rRNA sequence against corresponding region inGmorsitansmorsitansAmelliferaA glycine andT knoxi Dots indicate gaps introduced during alignment tomaximize homology UAAUhighlighted in blue forms the ldquohiddenbreakrdquo in rRNA sequences from different organisms

008

A glycine

An arabiensis

T tabaci

B mori

F arisanus

G m morsitans

A mellifera

96

99

100100

100

Figure 3 Amidpoint rooted maximum likelihood generated from 28S rRNA sequences for representative species in five major insect ordersThe cladogram was generated using PhyML with 100 bootstrap replicates Node labels show support bootstrap values

postprocessing of the rRNA which forms a UAAU tractdownstream of the 120573 segment of 28S rRNA Disintegrationof 28S rRNA molecule at the hidden break UAAU tract isinduced by heat denaturation that breaks hydrogen bondsjoining the 120572 and 120573 molecules to yield a single-bandedprofile for majority of insects [14] No specific buffer has

been reported to affect this dissociation Rather heatingof the RNA has been shown to yield similar results bothunder bioanalyzer analysis and on native agarose gels [14]Results of this study confirm presence of ldquohidden breakrdquoin majority of the insects and also reveal the absence of aldquohidden breakrdquo in the onion thrips Thrips tabaci Eight of


G

U

U

CG

C

G

A

G

10

U

G

A

GU

C

G

A

U

C

20C

U

AA

G

CC

U

A

A

30 A

G

A

G

A A

A

U

C

U

40U

U

A

G

U

A

U

G

G

U50

C

G

A

G

C

C

U

U

G

U

60A

G

G

U

UC

C

CA

(a) T knoxi

A

A

C

A

G

C

A

G

U10

U

G

G

A

C

AU

GG

G20

UU

AGU

C

G

A U

C

30

CU

AA

G

CC

AU

A

40

GG

G

A A

G

UU

CC

50

G

UU

U

C

A

A

A

G

G60

C

G

C

A

C

U

U

U

G

(b) A glycine

Figure 4 Continued


AA

CA

G

U

A

G

U

10U

G

C

U

C

GC

GA

G

20

UC

A

GU

CG

AUC

30C

UA

AG

CU

CA

A

40

G GA

GA

G A

U

CU

50

UA

U

GU

CG

AU

G60

UG

GC

G

UG

U

U

U

70

U U U U A U A

A U U

80

U UU

AAAAAUA

90

AUGAAAA

A

UA

100

ACG

AU rich tract

(c) B mori

Figure 4 Continued


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans

Figure 4 Comparison of proposed secondary structures for four insectsrsquo gap region of 28S rRNA sequences The AU rich tract that formsthe ldquohidden breakrdquo is indicated using an arc


the ten insects sampled here exhibit single banding profilesafter denaturation (Figure 1(a)) which supports earlier find-ings by Lehrach et al [4] andWinnebeck et al [14] Two clearbands (120572 and 120573 molecules) that are seen in case of whiteflies(Figure 1(a) Lane 3) appear to comigrate closely with the 18SrRNA molecule Their molecular size suggests dissociationof their 28S rRNA despite being in the same order withaphids This scenario is similar to that of testisrsquo rRNA fromrodents of genus Ctenomys which exhibits the presence ofldquohidden breakrdquo yielding a 26 Kb band and a 19 Kb band[15] This could mean that evolution has got no role to playin introduction of hidden break in insectrsquos RNA given thatwhiteflies and aphids are closely related evolutionarily

In addition to aphidsrsquo RNA (Figure 1(a) Lanes 1 and 2) anintact band for thripsrsquo 28S rRNA was observed which couldimply that this molecule does not contain a ldquohidden breakrdquo(Figure 1(a) Lanes 3 and 4) This observation was furtherstrengthened by comparative analysis of possible secondarystructures of variable regions that aligned to B morirsquos variableregion Structures predicted from T knoxi and A glycinedid not contain the AU rich region but rather showed highGC content of 47 and 49 respectively Neverthelessphylogenetic analysis of their sequences (Figure 3) did notsupport this observation Instead divergence was observedbetween their 28S rRNA sequences On the other handstructures of variable regions derived from G m morsitansand B mori displayed an AU rich tract and both showedlow GC composition (22 and 35 resp) In conclusionwe report absence of ldquohidden breakrdquo in one more insectthrips and suggest that the banding profile of all other insectsmay not always conform to a single band under denaturingconditions thus RNA profiling should not be used as ameansof characterizing newly discovered species

Conflict of Interests

Authors declare no competing interests

Acknowledgments

The authors wish to acknowledge DAAD for funding gradu-ate studies for Erick and Rosaline and Dr (s) Daniel MasigaHenri Muriuki Paul Mireji and Fathiya Khamis for editingthe paper andor for providing lab space

References

[1] A D Chapman Numbers of Living Species in Australia and theWorld Heritage 2nd edition 2009

[2] A V Kazantsev and N R Pace ldquoBacterial RNase P a new viewof an ancient enzymerdquo Nature Reviews Microbiology vol 4 no10 pp 729ndash740 2006

[3] P S Aranda DM Lajoie and C L Jorcyk ldquoBleach gel a simpleagarose gel for analyzing RNA qualityrdquo Electrophoresis vol 33no 2 pp 366ndash369 2012

[4] H Lehrach D Diamond J MWozney andH Boedtker ldquoRNAmolecular weight determinations by gel electrophoresis underdenaturing conditions a critical reexaminationrdquo Biochemistryvol 16 no 21 pp 4743ndash4751 1977

[5] H Ishikawa and R W Newburgh ldquoStudies of the thermalconversion of 28 S RNA of Galleria mellonella (L) to an 18 Sproductrdquo Journal of Molecular Biology vol 64 no 1 pp 135ndash144 1972

[6] K Ogino H Eda-Fujiwara H Fujiwara and H IshikawaldquoWhat causes the aphid 28S rRNA to lack the hidden breakrdquoJournal of Molecular Evolution vol 30 no 6 pp 509ndash513 1990

[7] H Fujiwara and H Ishikawa ldquoMolecular mechanism of intro-duction of the hidden break into the 28S rRNA of insectsimplication based on structural studiesrdquoNucleic Acids Researchvol 14 no 16 pp 6393ndash6401 1986

[8] Qiagen RNA Purification Qiagen RNeasy Micro HandbookProd Lit Qiagen 2003

[9] D A Benson I Karsch-Mizrachi D J Lipman J Ostell and EW Sayers ldquoGenBankrdquo Nucleic Acids Research vol 39 no 1 ppD32ndashD37 2011

[10] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[11] S Guindon F Lethiec P Duroux and O Gascuel ldquoPHYMLOnlinemdasha web server for fast maximum likelihood-basedphylogenetic inferencerdquo Nucleic Acids Research vol 33 no 2pp W557ndashW559 2005

[12] A Rambaut A Graphical Viewer of Phylogenetic Trees Instituteof Evolutionary Biology University of Edinburgh EdinburghScotland 2009

[13] AMWaterhouse J B Procter DM AMartinM Clamp andG J Barton ldquoJalview Version 2-A multiple sequence alignmenteditor and analysis workbenchrdquo Bioinformatics vol 25 no 9pp 1189ndash1191 2009

[14] E C Winnebeck C D Millar and G R Warman ldquoWhy doesinsect RNA look degradedrdquo Journal of Insect Science vol 10article 159 2010

[15] G J Melen C G Pesce M S Rossi and A R KornblihttldquoNovel processing in a mammalian nuclear 28S pre-rRNAtissue-specific elimination of an ldquointronrdquo bearing a hidden breaksiterdquo EMBO Journal vol 18 no 11 pp 3107ndash3118 1999

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contains a GC-rich hairpin loop that lacks the UAAU tract[5 7]

Efforts to understand molecular mechanisms involvedin the introduction of the gap have been hindered byunavailability of 28S rRNA secondary structures and highconservation of sequences reported in the regions that flankthe ldquohidden breakrdquo This makes it difficult to compare theldquohidden breaksrdquo [7] Nevertheless an earlier study suggestedthat the hidden break is probably introduced into the polynu-cleotide chain during or after the processing of 28S rRNAfrom its precursor [5]

In the present study banding profiles of RNA fromfive major insect orders considered of economic importanceincluding Diptera Hemiptera Thysanoptera Hymenopteraand Lepidoptera were compared with an aim of providingguidelines to the appearance of RNA for insects belonging tothese orders Importantly the absence of a hidden break wasobserved in the onion thripsThrips tabaci

2 Materials and Methods

21 Insect Sampling Representative insects for the fiveorders were obtained from insect colonies maintained atthe icipe insectaries and screen houses They included 150aphids (Hemiptera) Brevicoryne brassicae 60 mosquitoes(Diptera) Anopheles arabiensis 20 tsetse flies (Diptera)Glossina fuscipes fuscipes 16 honey bees (Hymenoptera)Apismellifera 200 whiteflies (Hemiptera) of Aleyrodidae familysix stem borers (Lepidoptera)Chilo partellus four silkworms(Lepidoptera) Bombyx mori 40 wasps (Hymenoptera)Fopius arisanus 10 fruit flies (Diptera) Dacus bivittatus and1000 thrips (Thysanoptera)Thrips tabaciThe sample sizewasinformed by the size of the fly and experiments were done inreplicates

22 RNA Extraction RNA from the insect samples wasextracted using the Direct-zol RNA Mini Prep Kit (ZymoResearch Corporation Irvin CA USA) following themanu-facturerrsquos instructions Fresh whole insect flies (le50mg) werehomogenized in 500 120583L of TRI-reagent and centrifuged inan Eppendorf AG 5417R centrifuge (Hamburg Germany) at12000timesg for 10min The supernatant was transferred intoa new tube followed by addition of one volume of absoluteethanol into the supernatant After vortexing 700 120583L of themixture was transferred into a Zymo-Spin IIC Column ina collection tube centrifuged at 12000timesg for 1min and theflow through discardedThe spin columnwas transferred intoa new collection tube and 400 120583L Direct-zol RNA prewashsolution was added and centrifuged at 12000timesg for 1minThe flow through was discarded and this step was repeatedInto the column 700 120583L RNA wash buffer was added andcentrifuged at 12000timesg for 1min The flow through wasdiscarded and the column spun again at 12000timesg for 2minto completely remove any residual wash buffer The columnwas then transferred into an RNase-free 15mL Eppendorftube and 25 120583L of DNaseRNase-free water was added ontothe column matrix centrifuged at maximum speed for 1min

for RNA elution The eluted RNA was immediately used fordownstream processes

23 Determination of RNAYield and Purity Thequantity andquality of the extracted RNA yield were determined using aNanodrop (ND) 2000c Spectrophotometer (Thermo Scien-tific Wilmington USA) by measuring optical density of 2120583Lof extracted RNA at A

260 nm and A280 nm respectively Any

sample with A260 nmA280 nm ratio below 17 was discarded

24 Denaturing Agarose Gel Electrophoresis A 12formaldehyde agarose (FA) gel was prepared by mixing12 g of agarose with 10mL of 10X FA gel buffer (200mMMOPS 50mM sodium acetate 10mM EDTA pH 70) Thiswas topped to 100mL using RNase-free water The mixturewas heated to melt the agarose and cooled to approximately65∘C after which 18mL of 37 123M formaldehyde and6 120583L of 10mgmL ethidium bromide were added Themixture was thoroughly but gently mixed then poured ontoa casting tray and left to polymerize under a laminar flowchemical hood

The RNA samples were mixed with 1 volume of 5XRNA loading dye prepared as per the Qiagen protocol [8]Sixteen 120583L of saturated aqueous bromophenol blue solutionwas mixed with 80120583L of 500mM EDTA of pH 80 720120583L of37 123M formaldehyde 2mL of 100 glycerol 3084mLformamide and 4mL of 10X FA gel buffer and topped up10mL with RNase-free water One volume of the dye wasadded to three volumes of RNA sample and mixed by pipet-ting The sample-dye mixture was down spun and incubatedat 65∘C for 5min then chilled on ice and immediately loadedonto the FA gel

The gel was run at 70V in the 1X FA gel runningbuffer (100mL of 10X FA gel buffer 20mL of 37 123Mformaldehyde and 880mL RNase-free water) for 1 hourfollowed by visualization of the gel under UV illuminationand analyzed using KODAK Gel Logic 200 Imaging System(Raytest GmbH Straubenhardt)

25 Nondenaturing Agarose Gel Electrophoresis A 12agarose gel was prepared by adding 12 g of agarose into100mL of 1X TAE buffer and heating to dissolve Six 120583L of10mgmL ethidium bromide was added to the mixture andthe sample RNA then mixed with a non-denaturing 6X DNAloading dye and the samples loaded onto the gel alongsidea molecular weight DNA marker (OrsquoGeneRuler 1 Kb plusDNA Ladder) Electrophoresis visualization and gel imageanalysis were carried out as outlined above

26 Multiple Sequence Alignment Phylogeny Reconstructionand Secondary Structure Determination Representative 28SrDNA sequences for the studied insect orders were obtainedfrom GenBank [9] (Table 2) In case of species lackingsequences from the database their sequences were substi-tuted with those of closely related species The 28S rDNAsequences of G fuscipes fuscipes and Brevicoryne brassicaewere substituted with G morsitans morsitans and A glycine







3 Results




GCcontent

Length(bp)






4 Discussion



M 1 2 3 4 5 6 7 8 9 10 1112 M

M 13 14 15 16 17 18 19 20 M

28S18S

5S

28S18S

5S

(a)

M 1 2 3 4 5 6 7 8 9 10 11

12

M

M 13 14 15 16 17 18 19 M

28S18S

5S

28S18S

5S

(b)


10 20 30 40 50 60 70 80 90 100 110






008

A glycine

An arabiensis

T tabaci

B mori

F arisanus

G m morsitans

A mellifera

96

99

100100

100





G

U

U

CG

C

G

A

G

10

U

G

A

GU

C

G

A

U

C

20C

U

AA

G

CC

U

A

A

30 A

G

A

G

A A

A

U

C

U

40U

U

A

G

U

A

U

G

G

U50

C

G

A

G

C

C

U

U

G

U

60A

G

G

U

UC

C

CA

(a) T knoxi

A

A

C

A

G

C

A

G

U10

U

G

G

A

C

AU

GG

G20

UU

AGU

C

G

A U

C

30

CU

AA

G

CC

AU

A

40

GG

G

A A

G

UU

CC

50

G

UU

U

C

A

A

A

G

G60

C

G

C

A

C

U

U

U

G

(b) A glycine

Figure 4 Continued


AA

CA

G

U

A

G

U

10U

G

C

U

C

GC

GA

G

20

UC

A

GU

CG

AUC

30C

UA

AG

CU

CA

A

40

G GA

GA

G A

U

CU

50

UA

U

GU

CG

AU

G60

UG

GC

G

UG

U

U

U

70

U U U U A U A

A U U

80

U UU

AAAAAUA

90

AUGAAAA

A

UA

100

ACG

AU rich tract

(c) B mori

Figure 4 Continued


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans







Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3 Results




GCcontent

Length(bp)






4 Discussion



M 1 2 3 4 5 6 7 8 9 10 1112 M

M 13 14 15 16 17 18 19 20 M

28S18S

5S

28S18S

5S

(a)

M 1 2 3 4 5 6 7 8 9 10 11

12

M

M 13 14 15 16 17 18 19 M

28S18S

5S

28S18S

5S

(b)


10 20 30 40 50 60 70 80 90 100 110






008

A glycine

An arabiensis

T tabaci

B mori

F arisanus

G m morsitans

A mellifera

96

99

100100

100





G

U

U

CG

C

G

A

G

10

U

G

A

GU

C

G

A

U

C

20C

U

AA

G

CC

U

A

A

30 A

G

A

G

A A

A

U

C

U

40U

U

A

G

U

A

U

G

G

U50

C

G

A

G

C

C

U

U

G

U

60A

G

G

U

UC

C

CA

(a) T knoxi

A

A

C

A

G

C

A

G

U10

U

G

G

A

C

AU

GG

G20

UU

AGU

C

G

A U

C

30

CU

AA

G

CC

AU

A

40

GG

G

A A

G

UU

CC

50

G

UU

U

C

A

A

A

G

G60

C

G

C

A

C

U

U

U

G

(b) A glycine

Figure 4 Continued


AA

CA

G

U

A

G

U

10U

G

C

U

C

GC

GA

G

20

UC

A

GU

CG

AUC

30C

UA

AG

CU

CA

A

40

G GA

GA

G A

U

CU

50

UA

U

GU

CG

AU

G60

UG

GC

G

UG

U

U

U

70

U U U U A U A

A U U

80

U UU

AAAAAUA

90

AUGAAAA

A

UA

100

ACG

AU rich tract

(c) B mori

Figure 4 Continued


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans







Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 3 4 5 6 7 8 9 10 1112 M

M 13 14 15 16 17 18 19 20 M

28S18S

5S

28S18S

5S

(a)

M 1 2 3 4 5 6 7 8 9 10 11

12

M

M 13 14 15 16 17 18 19 M

28S18S

5S

28S18S

5S

(b)


10 20 30 40 50 60 70 80 90 100 110






008

A glycine

An arabiensis

T tabaci

B mori

F arisanus

G m morsitans

A mellifera

96

99

100100

100





G

U

U

CG

C

G

A

G

10

U

G

A

GU

C

G

A

U

C

20C

U

AA

G

CC

U

A

A

30 A

G

A

G

A A

A

U

C

U

40U

U

A

G

U

A

U

G

G

U50

C

G

A

G

C

C

U

U

G

U

60A

G

G

U

UC

C

CA

(a) T knoxi

A

A

C

A

G

C

A

G

U10

U

G

G

A

C

AU

GG

G20

UU

AGU

C

G

A U

C

30

CU

AA

G

CC

AU

A

40

GG

G

A A

G

UU

CC

50

G

UU

U

C

A

A

A

G

G60

C

G

C

A

C

U

U

U

G

(b) A glycine

Figure 4 Continued


AA

CA

G

U

A

G

U

10U

G

C

U

C

GC

GA

G

20

UC

A

GU

CG

AUC

30C

UA

AG

CU

CA

A

40

G GA

GA

G A

U

CU

50

UA

U

GU

CG

AU

G60

UG

GC

G

UG

U

U

U

70

U U U U A U A

A U U

80

U UU

AAAAAUA

90

AUGAAAA

A

UA

100

ACG

AU rich tract

(c) B mori

Figure 4 Continued


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans







Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


G

U

U

CG

C

G

A

G

10

U

G

A

GU

C

G

A

U

C

20C

U

AA

G

CC

U

A

A

30 A

G

A

G

A A

A

U

C

U

40U

U

A

G

U

A

U

G

G

U50

C

G

A

G

C

C

U

U

G

U

60A

G

G

U

UC

C

CA

(a) T knoxi

A

A

C

A

G

C

A

G

U10

U

G

G

A

C

AU

GG

G20

UU

AGU

C

G

A U

C

30

CU

AA

G

CC

AU

A

40

GG

G

A A

G

UU

CC

50

G

UU

U

C

A

A

A

G

G60

C

G

C

A

C

U

U

U

G

(b) A glycine

Figure 4 Continued


AA

CA

G

U

A

G

U

10U

G

C

U

C

GC

GA

G

20

UC

A

GU

CG

AUC

30C

UA

AG

CU

CA

A

40

G GA

GA

G A

U

CU

50

UA

U

GU

CG

AU

G60

UG

GC

G

UG

U

U

U

70

U U U U A U A

A U U

80

U UU

AAAAAUA

90

AUGAAAA

A

UA

100

ACG

AU rich tract

(c) B mori

Figure 4 Continued


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans







Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AA

CA

G

U

A

G

U

10U

G

C

U

C

GC

GA

G

20

UC

A

GU

CG

AUC

30C

UA

AG

CU

CA

A

40

G GA

GA

G A

U

CU

50

UA

U

GU

CG

AU

G60

UG

GC

G

UG

U

U

U

70

U U U U A U A

A U U

80

U UU

AAAAAUA

90

AUGAAAA

A

UA

100

ACG

AU rich tract

(c) B mori

Figure 4 Continued


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans







Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AA

GG

C

GA

A

A

10

GC

U

G

A

A

A AUU

20UUCAAGU

U

A

U

30

A UAUAUAU

AA

40

AU

AA

UG

UA

UA

50

A

A

A

CAC

UU

GA

60A

AA

U

A

A

U

U

G

A

70

A

CG

AU rich tract

(d) G m morsitans







Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Acknowledgments


References























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article insects rna profiling reveals absence of...

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