RNAi phenotypes are influenced by the genetic background of the injected strain
© Kitzmann et al.; licensee BioMed Central Ltd. 2013
Received: 13 August 2012
Accepted: 19 December 2012
Published: 16 January 2013
RNA interference (RNAi) is a powerful tool to study gene function in organisms that are not amenable to classical forward genetics. Hence, together with the ease of comprehensively identifying genes by new generation sequencing, RNAi is expanding the scope of animal species and questions that can be addressed in terms of gene function. In the case of genetic mutants, the genetic background of the strains used is known to influence the phenotype while this has not been described for RNAi experiments.
Here we show in the red flour beetle Tribolium castaneum that RNAi against Tc-importin α1 leads to different phenotypes depending on the injected strain. We rule out off target effects and show that sequence divergence does not account for this difference. By quantitatively comparing phenotypes elicited by RNAi knockdown of four different genes we show that there is no general difference in RNAi sensitivity between these strains. Finally, we show that in case of Tc-importin α1 the difference depends on the maternal genotype.
These results show that in RNAi experiments strain specific differences have to be considered and that a proper documentation of the injected strain is required. This is especially important for the increasing number of emerging model organisms that are being functionally investigated using RNAi. In addition, our work shows that RNAi is suitable to systematically identify the differences in the gene regulatory networks present in populations of the same species, which will allow novel insights into the evolution of animal diversity.
For a long time, the identification of gene functions has been based on classical forward genetic screens where mutants are randomly generated, e.g. by chemical or transposon mediated mutagenesis. The established mutant strains are then screened for phenotypes and subsequently the disrupted gene is identified and further analyzed [1–5]. Importantly, it has been observed that the phenotypes of Drosophila and mouse mutants can depend on the genetic background of different strains, e.g. [6–13]. The same has been found for E.coli, rice and C.elegans [14–16]. In yeast, the portion of genes that are essential in only one of two closely related strains has been estimated to be about 6% . The unbiased forward genetic approach to identify gene functions has been very successful but it also limits the questions that can be addressed. First, saturating screens are only feasible in a very small number of model organisms [1–5, 18, 19]. Within insects, this is true only for the fruit fly Drosophila melanogaster while a few non-saturating screens have been performed in other insects including the red flour beetle Tribolium castaneum [20–22]. The limitation to highly developed model organisms at the same time limits the scope of biological questions that can be asked. A further restriction of forward genetics is that mutant strains need to be maintained over time, which represents a significant effort feasible only with the support of large scientific communities. Moreover, the genetic tools, which facilitate stock keeping (e.g. balancer chromosomes) are not available in most organisms and are tedious to construct.
The discovery of RNA interference (RNAi) in animals  has opened the possibility to study gene function in many more animals and has significantly contributed to a an expansion of biological questions that are studied in terms of gene function. In RNAi, double stranded RNA (dsRNA) within cells is processed by the highly conserved RNAi machinery including the Dicer protein, which cuts the long dsRNA into 21mers. These are loaded into the destruction complex (RISC complex), which is guided by the single stranded small interfering RNAs (siRNAs) to mRNAs with complementary sequence. The Argonaute protein as part of the RISC complex eventually cuts the mRNAs within the region of complementary, leading to the destruction of the mRNA and consequently to a reduction of the gene product [24–26]. RNAi is an anti-viral defense system, is required for the silencing of transposons  and highly related processes act in post-transcriptional gene regulation, the control of chromatin and RNA polymerase II transcription elongation activity [24, 28].
The RNAi response of some organisms is systemic, i.e. dsRNA delivered into the body cavity is distributed throughout the organism and enters all cells. Hence, local injection leads to systemic gene silencing [24, 26, 29, 30]. In some organisms like C. elegans and Tribolium the RNAi effect is transmitted even from injected parents to the offspring [24, 29–32].
RNAi in the red flour beetle Tribolium castaneum is robust, systemic, splice variant specific and feasible at all developmental stages [31–35]. Moreover, it is able to phenocopy genetic Null alleles at least in some instances, e.g. in the case of Tc dfd , Tc distal less [32, 36]Tc krüppel  and Tc knirps (Bucher, unpublished). The strength of the RNAi response can be experimentally modulated by varying the concentration of injected dsRNA or by varying the time between injection of the mother and collection of the phenotypic offspring [32, 34, 38–40].
In an ongoing genome wide RNAi screen in Tribolium (iBeetle screen, unpublished), females of the black strain  were injected with dsRNA of the fragment iB_00198 and were subsequently mated to pig19 males . In the cuticle of offspring first instar larvae, specific labrum defects were observed with high frequency. The knocked-down gene product is an Importin α, which belongs to the karyopherin multi-gene family of nuclear import receptors . In metazoans three classes of importin α genes exist: importin α1, importin α2, and importin α3 . Importin α proteins are nuclear import adaptors, which bind cargos containing a classical nuclear localization signal (cNLS) sequence . The Importin α-cargo heterodimer forms a trimeric complex with the actual importer Importin β, which enables the passage of the cargo through the nuclear pore complex . The Importin α1 protein shows a tandem array of ten armadillo (ARM) repeats, where the ARM domains 1 to 4 (major site) and the domains 4 to 8 (minor site) are responsible for recognition and binding of specific cargoes [47, 48]. All members of the importin α family function the same way and it has been shown that they act redundantly on many cargoes but there are also cargoes, which require a specific Importin α for their nuclear import [44, 45, 49–54]. In the yeast S. cerevisiae, estimated 57% of steady-state nuclear proteins use this import system . Considering this, it was surprising that the knock-down of a gene which encodes such a widely required factor would lead to such a specific cuticle phenotype in Tribolium.
In this work, we quantitatively compare the RNAi phenotypes of Tc-importin α1 in two Tribolium laboratory strains, black and San Bernadino (SB). Surprisingly, we find that RNAi knock-down leads to qualitatively different phenotypes depending on the strain. Further, we show that this is neither due to a general difference in RNAi sensitivity of these strains nor to nucleotide sequence divergence between them or differential embryonic expression. Instead, we find that the genotype of the injected female determines the RNAi phenotype of the offspring. These results show that the phenotypes generated in RNAi experiments can depend on the genotype of the used strain and we suggest that a proper documentation of the strain is an essential piece of information when publishing RNAi studies in any species.
Results and discussion
iB_00198 dsRNA targets Tc-importin α1
Tc-importin α1 pRNAi cuticle phenotype is different in the two strains
In order to test whether this unexpected phenotypic difference was due to the selection of dsRNA fragments different from the one used in the screen (Additional file 1A and B), or alternatively, from the use of a different strain, Tc-importin α1 RNAi was repeated in the black strain. Both non-overlapping dsRNA fragments (1 μg/μl) were injected into black female pupae, which were mated with black males (Figure 2F-J) or pig19 males (i.e. the combination used in the screen; Additional file 1 D). The knock-down using the Tc-importin α1a dsRNA (Figure 2F, n=22) frequently resulted in cuticles with an affected labrum (86.4%) which was either deformed (36.4%; Figure 2H) or completely absent (50%; Figure 2I, J). In a portion of the cuticles, other head defects (antennal and gnathal: 22.7%) or abdominal defects were found (4.5%). Notably, the “labrum only” phenotype was frequent (>60%). These observations were confirmed using the non-overlapping fragment with the only difference that additional dorsal cuticle defects were observed (28.6%; Figure 2G). To further confirm our finding, we repeated the RNAi with the original iB_000198 dsRNA fragment (1 μg/μl) in the black and SB strains, which resulted essentially in the same strain specific phenotypes (not shown; the original documentation of all Tc-importin α1 RNAi experiments is found in Additional file 1 E).
Taken together, these results showed that the knock-down of Tc importin α1 led to different phenotypes depending on which strain was injected and that this difference was not due to off target effects. Because both non-overlapping dsRNA fragments resulted in similar phenotypes, the following experiments were done using the Tc-importin α1a fragment. Importin α proteins are essential parts of the nuclear import machinery and have housekeeping functions . Therefore, one would expect a dramatic and pleiotropic loss of function phenotype. The phenotype in the SB strain matches this expectation pretty well. Also in the black strain, some pleiotropic defects are observed, which increase somewhat in number at higher dsRNA concentrations. This is an indication that the expected pleiotropic phenotype is present but strongly reduced in the black strain.
The phenotypes are qualitatively different
Injecting lower amounts of dsRNA (0.3 μg/μl) into the SB strain led to a decreased frequency of cuticular defects, while their quality was similar to the 1 μg/μl experiment (compare Figure 4A with B). Specifically, the “labrum only” phenotype was not found. Using 3 μg/μl dsRNA led to an “empty egg” phenotype in all animals. Empty egg phenotypes are an indicator of very severe embryonic defects leading to the abortion of embryonic development prior to cuticle secretion resulting in empty egg shells in cuticle preparations (Figure 4C).
Knock-down of Tc-importin α1 in the black strain using 0.3 μg/μl dsRNA resulted in a very similar phenotypic pattern as shown for 1 μg/μl (compare Figure 4D with E). Notably, “labrum only” phenotypes were found in about 80% (Figure 4D) where the labrum was absent in 41.2%. Other defects were observed with low frequency. Injection of 3 μg/μl dsRNA led to cuticles with a comparable occurrence of labrum defects but with a slightly increased portion of other cuticular defects (Figure 4E, F). As consequence, the number of “labrum only” (i.e. labrum but no other structure affected) phenotypes dropped to 30% but the labrum remained the most frequently deleted structure (40%). Finally, we tested the effect of Tc-importin α1 RNAi in two other strains. We injected females of the pig19 strain (derived from the pearl genetic background) and mated them with black males. The phenotype of the offspring was intermediate between black and SB injected females (Additional file 1I). In the Georgia-2 (GA-2) genetic background, the injected females became sterile not allowing judging the cuticle phenotype of the offspring (Additional file 1 J).
In summary, the phenotypic series generated by RNAi with the same dsRNA in different strains differed qualitatively in several respects: “Labrum only” phenotypes were found exclusively in the black strain while the abdomen inside-out phenotype always remained below 5%. Moreover, increasing dsRNA concentrations led to a rather mild increase of phenotypic severity but even at highest concentrations the “empty egg” phenotype was not increased beyond background. In the SB strain, in contrast, “labrum only” phenotypes were not found at all, while the abdomen inside out class was always high. Increasing amounts of dsRNA led to a significant increase of phenotypic severity leading to 100%“empty egg” phenotypes at high concentrations. At the same time, the phenotypes also show similarities: The pleiotropic defects seen in SB and to a minor extent also in black represent the expected pleiotropic phenotype of a nuclear import protein.
RNAi sensitivity is similar in the black and SB strains
The different RNAi phenotypes could be due to a different strength of the RNAi response in these strains or alternatively could be due to the different genetic background, which interacted differently with Tc-importin α1 but not other genes. To test this, we first quantified the transcript level in the RNAi animals by qPCR. The expression was reduced by >93% in both strains in both 0-2h, 10-12h egg collections as well as in ovaries (Additional file 1 K). As a complementary means to compare RNAi efficiency in the strains, we quantitatively compared the phenotypic range induced in the SB and black strains after RNAi using the same dsRNA preparations targeting four different genes.
The analogous experiment was performed with Tc-gt dsRNA (1 μg/μl, n=50). The number of deleted trunk segments was used as measure for the phenotypic strength. This number varied from zero to six deleted trunk segments (Figure 5B). Overall, the distribution in both strains was similar, while both the strongest and the mildest phenotypes were only observed in SB cuticles (Figure 5B, gray bars). Also the average of the number of deleted segments was very similar (ø black: 2.30, ø SB: 2.32).
Further, we performed larval RNAi for Tc-wingless (Tc-wg) and Tc-six3 in order to compare the RNAi response after larval RNAi (lRNAi). Knock-down of Tc-wg via injection of dsRNA (1 μg/μl) into late larval stages (L6) of SB and black lead to pupae with reduced genital lobes, an increased distance between the pupal wings and a reduced maxillary diameter (Figure 5C). The latter was the best quantifiable indicator because the diameter is very constant in wt pupae (Additional file 1 G). The phenotypic series was divided into four categories (Figure 5C, panel 1–4), was rated and the average was calculated. Both strains show a comparable mean value (ø black: 2.9, ø SB: 2.8).
Tc-six3 dsRNAi injection (0.5 μg/μl) in L6 larvae led to pupae with reduced eye size (Figure 5D), which was quantified (see experimental procedures). Again, the phenotypes were grouped into four categories and the mean values were calculated (Figure 5D). Tc-six3 lRNAi resulted in slightly stronger pupal phenotypes in the SB strain (ø black: 2.8, ø SB: 3.1).
Taking into account the experimental variability inherent to RNAi experiments, these data suggest that our strains do not have a generally different RNAi response. Moreover, the nucleic acid sequence of the dsRNA fragments is almost identical in both strains (99.4%), making different RNAi efficiencies due to mismatches unlikely. Hence, the strain specific phenotypic difference we observed was likely due to different modulation of the Tc-importin α1 phenotype in the respective genetic backgrounds. This is similar to findings in other model organisms, where the phenotype of mutant alleles of some (but not all) genes is different depending on the genetic background of the strain.
Tc-importin α1 peptide sequence is slightly diverged
Next, we asked whether differences of the amino acid composition of Tc-importin α1 protein could be the reason for the differences. We isolated and sequenced the coding sequences of both strains. The Tc-importin α1 amino acid sequences (526 amino acids) of the black and SB strains were aligned with the sequenced strain (GA-2) and with Importin α orthologs of other species.
The second different amino acid is located within the eighth ARM domain (position 376). Here, the SB strain encodes a leucine (L, hydrophobic, not polar), whereas the two other Tribolium strains show a glutamine (Q, polar uncharged), which is also found in the two hymenopterans Nasonia vitripennis (N. vitripennis) and Apis mellifera (A. mellifera). M. musculus carries an alanine (A) at this position, which is hydrophobic and not polar, whereas D. melanogaster has a glutamic acid, which is acidic and negatively charged. None of the two sites is predicted to be the target of phosphorylation, N-glycosalation nor N-myristoylation by ExPASy and Prosite analysis. Taken together, the observed amino acid substitutions may lead to altered binding affinities, which might influence the phenotype. However, this is difficult to test because we do not know which of the many nuclear proteins likely to be imported by Importin αs actually elicit the observed phenotypes. An alternative explanation would be that the mutations lead to differential splicing of the gene in the different strains.
The maternal genotype mainly determines the Tc-importin α1 phenotype
Our data hinted at the genetic background as cause for the different phenotypes. However, it remained open whether this would be based on zygotic gene expression (i.e. expression of the embryonic genome) or whether different maternal inputs would be involved (e.g. differential loading of the egg with respective protein or mRNA). In order to distinguish between these possibilities, Tc-importin α1 dsRNA (1 μg/μl) was injected in SB and black females and these were afterwards mated with males of the other strain, respectively. Therefore, zygotic expression of genes was based on heterozygous condition for black/SB in all offspring while the maternal contribution was either of the black or the SB type.
These results show that it is primarily the genotype of the mother, which determines the quality of the Tc-importin α1 RNAi phenotype while the minor increase of “labrum only” phenotypes to 4,3% indicate some influence of the zygotic genome, too.
The importance of maternal contribution of Importin α proteins is plausible, because it is known from Drosophila that the transition from maternal to zygotic control occurs only at cell cycle 13–14 [55, 56]. Already before this transition the nuclear import machinery is essential to allow gene activity. This is ensured by a strong maternal contribution [57–59].
Relative maternal contribution of Tc-importin α1 is reduced in the black strain
Based on the fact that Importin α proteins act redundantly in the import of most proteins [43, 46] we asked whether the maternal supply of the oocyte with importin mRNAs would be different in the SB and black strains. Specifically, we wanted to test the model that the contribution of maternal Tc importin α1 was relatively small in the black strain and that this was buffered by increased Tc importin α2/3 contributions. In that case, knock-down of Tc importin α1 would lead to less prominent phenotypes in the black strain because most defects would be buffered by the other Importins α’s.
Maternal increase of Tc-importin α2 in Tc-importin α1 RNAi embryos in the black strain
An alternative possibility for different maternal buffering of the Tc-importin α paralogs was that the knock-down of Tc-importin α1 would be compensated by different patterns of upregulation of the other Tc-importin α paralogs in these strains. Indeed, in 0-2h old embryos of Tc-importin α1 RNAi animals, the load of Tc-importin α2 was significantly increased in the black but not the SB strain (see Additional file 1 K). At this embryonic stage, maternal messages predominate, hence, it appears that maternal upregulation was involved in rescuing parts of the phenotype in the black strain. Interestingly, the situation was different when measuring zygotic transcript levels in 10-12h embryos. Here, the black transcript levels are not altered much, while in SB, Tc-importin α2 and 3 are upregulated. Apparently, the delayed compensation in SB is too late to rescue the embryonic phenotype.
Potential mechanisms leading to the phenotypic differences
This work was primarily aimed at showing that the quality of RNAi phenotypes may depend on the genetic background of the strain used. However, we also gained some insights into the potential mechanism how the difference might arise in the specific case of Tc importin α1. Two things need to be explained: First, the absence of the expected pleiotropic defect, and second the occurrence of the qualitatively different “labrum only” phenotype in the black strain. To explain the apparent decrease of pleiotropic defects, we suggest that females of the black strain load their oocytes with more Tc importin α2 and 3 relative to Tc importin α1 and moreover, compensate for loss of Tc importin α1 by upregulating Tc importin α2. Therefore, the importin paralogs are able to rescue the knock-down effect of Tc importin α1 much better in the black than in the SB strain. This would lead to a comparably mild pleiotropic phenotype. Indeed, functional redundancies  and the resulting masking of phenotypes  by the different Importin α’s was described previously in Drosophila.
The black specific “labrum only” phenotype might depend on the strain specific amino acid changes found within the cargo binding domains. This difference may have allowed one or several target proteins required for labrum development to evolve an Importin α1 specific import signal. Loss of Tc-Importin α1 would be compensated by the paralogs for most proteins but not for the labrum specific protein, leading to the observed labrum specific phenotype. This model is in line with data showing that Importin paralogs besides their redundant roles in nuclear import of many proteins do also have paralog-specific cargoes [54, 62]. A prerequisite for testing this model is the identification of all genes that lead to “no labrum” phenotypes. Then, these could be tested for differential binding with the different Importin αs.
Alternatively, a higher relative expression of Tc-importin α1 in the labrum anlagen of the black strain could contribute to a “labrum only” phenotype. However, we were not able to detect differences in labral expression of Tc-importin α1 in the black and SB strains by in situ hybridization. We might have missed mild modulations of expression–whether such minor differences would be able to lead to such a clear phenotype remains questionable. However, with the current data, we cannot exclude that the mechanism is much more complex and may involve many additional factors and interactions.
Documentation of strains is essential for future RNAi studies
It has been known from mice and Drosophila that different genetic backgrounds of laboratory inbred strains can affect the phenotypes in transgenic experiments (e.g. [6–8, 63–65]. This may be due to changes within coding or non-coding regions . Recently, Dworkin et al. argued that strain specific modulation of phenotypes may have to be considered more systematically than in the past . Here, we show that this is also true for RNAi studies, which to our knowledge has not been considered in the past. Our findings have implications for the increasing number of RNAi experiments in an increasing number of animal taxa. First, discrepancies of results between labs might be due to the use of different strains. Second, the strain used in an RNAi experiment needs to be documented and kept over time to allow the reproduction of the phenotypes by others. Third, confirming the results of an RNAi experiment in another strain provides a good means to test for the general relevance of a phenotype.
RNAi as tool for studying genetic differences on the population level
Our finding also opens new possibilities. The ease of application of RNAi allows systematically identifying differences in gene regulatory networks between populations of one species including species that cannot be kept in the lab. Such changes provide the genetic variability, which is required for the evolution of novel traits. RNAi will allow a systematic investigation of the degree of variability within species.
Tc-importin α1 open reading frame sequence (1581 bp; accession: [XM_963412]) was obtained from the iBeetle genome browser (http://bioinf.uni-greifswald.de/gb2/gbrowse/tcas/). The following primers were used to amplify the open reading frame froman embryonic cDNA pool (0-48 h) via standard PCR: 5’-ATGTCGGGCTCCGCTCACAA-3’ and 5’-TTAAAAATGGAATCCTCCCATCGGCACCG -3’. The Tc-importin α1 open reading frame was cloned into the pJET1.2 vector.
The sequences of the fragments used for RNAi are given in the Additional file1 B. The templates for the non-overlapping fragments were generated by PCR from a plasmid template using following primers: 5’-TAATACGACTCACTATAGGAGTCTGGAGGAGGGTTCTTGC-3’ and T7 Primer (5’-TAATACGACTCACTATAGG-3’) for the 5’ fragment (Tc importin α1 a, 709 bp; see Additional file 1 A and B, gray bar) and 5’-TAATACGACTCACTATAGGTTGCGAAAGTCTCCCCAGCT-3’ and pJET1.2R sequencing primer with a T7-attachment (5’-TAATACGACTCACTATAGGAAGAACATCGATTTTCCATGGCAG-3’) for the 3’ fragment (Tc importin α1 b, 872 bp; see Additional file 1 A and B, black bar). Concentrations for parental RNAi were 0.3 μg/μl, 1 μg/μl and 3 μg/μl (Tc importin α1 a and Tc importin α1 b), 1 μg/μl (Tc gt and Tc dll) and for larval RNAi 1 μg/μl (Tc wg) and 0.5 μg/μl (Tc six3). Pupal injections were performed as described . For larval RNAi (lRNAi) the larvae were anaesthetized by cooling them on ice. The dsRNA was injected using the FemtoJet express (eppendorf, Hamburg). Late larval stages (L6) were injected into the ventro-lateral side between the fifth and sixth abdominal segment. On average 0.4-0.5 μl dsRNA were injected into one larva. Injected larvae were raised as described .
Microscopy and Image analysis
Cuticles were documented using a Zeiss LSM 510 as described [68, 69]. Tc dll RNAi legs were recorded in 15 focal planes using a Zeiss Axioplan microscope and Image-Pro Plus software (MediaCybernetics®, version 6.2). Deconvolution was performed with the “No Neighbour” method followed by a maximum projection using ImageJ (version 1.44 o). Pupae were analyzed and documented using a Leica M205 FA fluorescence stereomicroscope.
lRNAi pupae were analyzed using ImageJ (version 1.44 o). The diameter of the second segment of the maxillary palpus (Figure 5C, white arrowhead) was measured using the straight line tool. Division in phenotypic levels: Level 1= wild-type (wt) range of diameter, level 2= minimum wt diameter minus wt range, level 3= minimum level 2 diameter minus wt range, level 4= minimum level 3 diameter minus wt range. The eye field area of both eyes in Tc-six3 lRNAi in late pupal stages (fully sclerotized mandibles) was measured by freehand selection tool and the mean was calculated. The mean value provided the basis for the phenotype comparison. The phenotype level was chosen arbitrarily.
Importins of the different species were obtained using amino acid sequence of Tc-Importin α1 as query for a BLASTp search  at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi, Figure S2). Phylogenetic analysis was conducted using MEGA version 5 . The multiple alignment was done using the ClustalW application with the preset parameters. A phylogenetic tree was calculated using the Neighbor-joining method under the Poisson amino acid substitution model. Bootstrap analysis was conducted using 1000 replicates to test the robustness of the phylogenetic tree. Calculation of a phylogenetic tree using the Maximum-Likelihood method under the Jones-Taylor-Thornton model amino acid substitution model results in essentially the same phylogenetic tree.
For correspondence analysis , the labrum, head bristle pattern, antennae, gnathal appendages, thoracal segments, abdominal segments, pygopods and urogomphi of each L1 cuticle were classified into three different categories: not affected=0, deformed=0.5 and absent=1. The dataset was imported into R (v. 2.14.2, ) and correspondence analysis and plotting was performed by using the R ‘ca’ package .
Total RNA was isolated from dissected ovaries of adult beetles using the Tissue & Insect RNA MicroPrep™ Kit (Zymo Research Corporation, Irvine) and from eggs (0–2 h and 10–12 h) using TRIzol® reagent (Ambion®/Live technologies, New York). 1 μg/μl total RNA was converted to cDNA by using the MAXIMA® First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, Waltham). Quantitative PCR was performed using HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) (Solis BioDyne, Tartu) and the CFX96™ Real-Time PCR System (Bio-Rad Laboratories, Hercules). For the qRT-PCR the following primer pairs were used: Tc-importin α1: 5’-CCGTATGCTGTGCTAATCGAG-3’ and 5’-CGTCCCGAAGAAGTGTTCAAT-3’, Tc-importin α2: 5’-AAAGTCTACGAACGGGCTTTG-3’ and 5’-GAACTGAATCTCCCCATTTGC-3’, Tc-importin α3: 5’-TGAGGAGTGCAATGGCTTAGA-3’ and 5’-TCATCCGCATCACCACTAAAG-3’ and Tc-rpS3: 5’-ACCTCGATACACCATAGCAAGC-3’ and 5’-ACCGTCGTATTCGTGAATTGAC-3’. All primers were designed to span an intronic sequence and were validated by gel analysis. To calculate primer efficiency (E=10^(−1/m)), a dilution series was performed. Data was normalized by the formula: rel. expression=Rtarget^Cqtarget/Rref.^Cqref. For better comparison of the results in the two strains, the expression levels were normalized such that the genes with the lowest relative expression were set to the same values in the SB and black strains. Specifically, the difference between the means of Tc-importin α1 expression levels in the SB and the black strains were calculated. Subsequently, all SB expression levels were reduced by this mean difference (Figure 8). This normalization was not done for Additional file 1 K. For statistical analysis, three tests were performed: Welch’s t-test, Student’s t-Test and the Mann–Whitney-U-test. None of these tests revealed significant differences between the respective expression profiles in SB and black.
We thank the DFG research unit FOR1234 for funding and the members of the iBeetle consortium for discussion. We thank Sebastian Kittelmann for critically reading the manuscript, Nico Posnien and Georg Oberhofer for help with the statistical analysis and qPCR and Ernst A. Wimmer for continuous support.
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