Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.
The complete protocol with different steps is depicted in the flow chart in Figure 1. Individual steps constituting dissection, RNA extraction, First Strand cDNA synthesis and qRT-PCR are detailed below.
I. Larval Dissection
II. RNA Extraction (As per manufacturer s protocol for using Trizol Reagent)
Homogenization
Phase separation:
RNA precipitation
RNA Wash
RNA elution
III. First-Strand cDNA Synthesis (As per manufacturer s protocol using SuperScript first strand Synthesis kit by Invitrogen.)
RNA | x μl |
10mM dNTP mix M | 1μl |
Oligo(dt) (0.5 μg/ μl) | 1 μl |
DEPC treated water | (8-x) μl |
Total Volume: | 10 μl |
Note: x is the volume of RNA used for cDNA synthesis. Depending on the concentration of the RNA, 2-3 μg of RNA will be used for each reaction and the total volume of each reaction should be 10 μl. |
10X RT Buffer | 2μl |
25mM MgCl2 | 4 μl |
0.1M DTT | 2 μl |
RNase out | 1 μl |
IV. qRT-PCR:
Primer Design and Determination of reference gene
Preparation of standards
Plate Template
Set up the cycling parameters
Plate set up for CFX96 (BioRad) machine
Nuclease free water | 2μl |
2.5 X SYBR green | 4 μl |
Primer forward | 1 μl |
Primer reverse | 1 μl |
Total volume | 8 μl |
V. Diagrammatic Representation of the Experiment
Figure 1: Flow chart depicting the sequential order of steps for the gene expression study.
Figure 2: A) larval EAB dissection showing midgut in the middle. B) isolated midgut of larval EAB
Figure 3: A) Mean relative expression values (REVs) for a peritrophin gene (AP-PERI1) in different larval tissues including cuticle (Cu), midgut (MG) and fat bodies (FB). An EAB specific ribosomal protein (herein named, AP-RP1) was used as the internal control for normalizing the data obtained for the gene of interest, AP-PERI1. B) Relative fold change of AP-PERI1 in larval tissues. The cuticle tissue showed the least expression and therefore was taken as the calibrator (1X) sample (Pfaffl, 2001).
Figure 4: A) Mean relative expression values (REVs) for a peritrophin gene (AP-PERI1) in different developmental stages of EAB including larval instars (1st, 2nd, 3rd and 4th), prepupae (PP) and adults (A). An EAB specific ribosomal protein (AP-RP1) was used as the internal control for normalizing the data obtained for the gene of interest, AP-PERI1. B) Relative fold change of AP-PERI1 in various developmental stages. The PP sample showed the least level of mRNA levels and therefore was taken as the calibrator (1X sample).
VI. Conclusion
Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. Further, qRT-PCR has mostly been the key tool to validate data generated from high throughput gene expression analyses such as microarrays and the new-generation RNA-Seq.
The threshold cycles (Ct) value is obtained for each sample in the qRT-PCR plate. For making the standard curve, the Ct value obtained for each dilution was plotted against the log of its concentration. The Ct values for the experimental samples were then plotted onto this dilution series standard curve. Target quantities were calculated from separate standard curves generated for each experiment i.e. tissue and developmental expression. The relative expression values (REVs) were then determined by dividing the quantities of the target sequence of interest (in this case AP-PERI1) with the quantity obtained for the internal control (AP-RP1). For calculations of significance, the logs of the REVs for each gene were analyzed by ANOVA (Analysis of Variance) using the PROC MIXED procedure of SAS (SAS Institute Inc. SAS/STAT User s Guide, Version 9.1). Significance in expression level was determined at p < 0.05. Relative fold changes in the case of tissues were calculated by taking the cuticle sample as the calibrator (1X sample, Pfaffl, 2001). Therefore, the expression levels in the midgut and fat bodies were relative to the cuticle. In the case of developmental expression, the prepupal sample was taken as the calibrator. For both studies, two biological replicates and three technical replicates were used.
Our experiment revealed significantly (p < 0.05) higher levels of AP-PERI1 transcript in the midgut tissue (Fig. 3) compared to other tissues assayed. During development the feeding stages (i.e. the larval instars and adult) were significantly (p < 0.05) higher than the prepupae sample (Fig. 4). The least mRNA levels were calculated for cuticle and prepupae (Figs. 3 & 4). Given that peritrophins are integral component of the insect midgut our results corroborate previous findings in other insect species (Mittapalli et al. 2007). The patterns observed during the developmental stages further confirm the function of AP-PER1 in digestion and -related processes. The widely used Bacillus thuringiensis (Bt) toxins in crop plants indeed target and disrupt the PM in insects (Pauchet et al., 2009). Hence, the results obtained in this study could not only reveal potential control targets for EAB but also provide fundamental knowledge as to how invasive insect species adapt to their new environments.
The authors have nothing to disclose.
We acknowledge the help provided by Lourdes Delta Arrueta Antequera (Department of Entomology, Ohio State University/OARDC, Wooster, OH) in the setup of the experiments. We thank Dr. Therese Poland (USDA, Forest Services, NRS) for send EAB larval samples. Help provided by Dr. Luis A Canas and Nuris M Acosta with the microscope setup is appreciated. Funding for this project was provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.