We have also been able to detect expression of this receptor in all studied tissues, which is consistent with the pleiotropic nature of growth hormone in fish (20–23). The expression level of tiGHR that we obtained for each studied tissue can be organized in decreased order of expression levels as: liver > muscle > brain > heart > gonads > intestine > stomach > spleen (Fig. 6). The highest expression level of tiGHR I in liver is consistent with previous receptor binding studies (20). Besides, it is in agreement with previous conventional RT-PCR studies (4, 6) and with real-time PCR studies (24). As it is expected, the tissue distribution obtained for us is more similar to the real-time RT-PCR results than to the results of the other studies using conventional RT-PCR. The fact that we observed statistically significant differences only between liver–spleen and between liver–stomachs is due to a non-parametric test that we used. These tests are less powerful than the parametric tests that assume data Gaussian distributions. With small samples (n = 3), non-parametric tests have little power to detect differences especially when we work with biological samples that are intrinsically variable. In future experiments, we will work with a higher number of animals. To refine our initially found results, we would retest them with a higher resolution of competitor molecules because we demonstrate in the validation experiments that the coefficients of variability are lower when the molecule numbers of target and competitor sequences in the samples are similar.
Despite the fact that the described competitive RT-PCR assay is labor intensive, less sensitive, and has a lower dynamic range than the real-time assays, it is less expensive than the real-time RT-PCR studies.
In summary, through this work, we have developed a quantitative RT-PCR assay by competition that was sensitive enough to differentiate among mRNA abundance levels of tiGHR I. The nature of competition reactions observed was supportive to prove the authenticity of quantification of tiGHR I.
Acknowledgments This research was funded by the Center for Genetic Engineering and Biotechnology. The authors would like to thank Dr. Ricardo L. Lleonart for his valuable assistance and review of this manuscript and MSc. Yoelys Cruz for her advice in statistical processing of data.
Appendix
PROTOCOLS
| I- Cloning of competitor Materials• RNAgents: Total RNA Isolation System,Promega Z5110• PolyATract: mRNA Isolation System II, Promega Z5200• Reverse Transcription System, Promega A3500• PCR Master Mix, Promega M7505• QIAquick Gel Extraction Kit, QIAGEN 28704• pGEM®-T Easy Vector System I, Promega A1360 and pBluescript® II Phagemid Vectors, Stratagene 212207 to cloning DNA sequences• Reagents for cloning• Ampicillin and streptomycin for selection purposes• Degenerated primers to amplify tiGHR I probe sequence• Top 10 (F− mcrA Δ[mrr-hsdRMS-mcrBC] ø80 lacZ Δ M15 Δ lacX74deoR recA1 araD139 Δ[ara-leu] 7697 galV galK rpsL [StrR] endA1nupG) or equivalent electrocompetent E. coli cells• T7 RiboMAX TM Express RNAi System, Promega P1700• MEGAscript® RNAi Kit, Ambion 1626 Methods1. To obtain total RNA of tilapia (O. niloticus) liver, we followed the procedure described in the section IV of the Technical Bulletin 087 (TB087) of RNAgents® Total RNA Isolation System (Promega). We started with 1 g of tilapia liver.2. Starting with 5 mg of tilapia liver total RNA, we obtained mRNA following exactly the protocol described in the section IV in the Technical Manual 021 (TM 021, Promega) to PolyAtract® Systems I.3. RT-PCR amplifies the tiGHR I probe. The reverse transcription reaction was performed with 1 µg of tilapia liver mRNA and Oligo (dT)15 as primer following the protocol described in the section III of the Technical Bulletin 099 (TB099) of Reverse Transcription System (Promega, USA). We used 10 µl of the five times diluted RT reaction in 50 µl of the PCR final, volume. We used too 25 µl of PCR Master Mix 2× (Promega) and 3 µM of each degenerated primer (150 pmol/50 µl PCR). We performed 3 min to 95°C to denaturalize all DNAs in the reaction and after we did cycling program (1 min to 94°C, 1 min to 42°C, and 1 min to 72°C) for 30 cycles and a final extension 5 min to 72°C.4. Clone the PCR product nearby to 500 bp into the pGEM-T Easy, or equivalent, vector and transform into electrocompetent top ten E. coli cells, or equivalent, for sequence verification.5. Sub-clone the 3′ region of the tiGHR I probe into pBS KS + vector and ensemble again duplicating an internal Pst I–Acc I fragment (Fig. 1 a).6. Linearize the plasmid that contains the competitor sequence under the control of T7 RNA polymerase promoter with appropriated restriction enzyme.7. Synthesize single-stranded transcript of competitor RNA. We followed the protocol described in the section III-C of the Technical Bulletin 316 of the T7 RiboMax™ Express RNAi System (Promega)8. Remove the DNA template by digestion with RNase-free DNase of the T7 RiboMax™ Express RNAi System.9. Purification of ssRNA was carried out following the protocol described in the section III-E of the Instruction Manual of the MEGAscript® RNAi Kit (Ambion, USA).10. Quantitate the product by measuring its absorbance at 260 nm and examine the integrity on a 1% denaturing agarose gel.11. Store RNA competitor precipitated in EtOH at −20°C. | |
| II. Validation of Competitive PCR Materials• Linear DNA of target and competitor sequences.• Gene-specific primers to amplify target and competitor sequence.• PCR Mas, ter Mix, Promega M7505• Reagents and equipment to do DNA electrophoresis.• Digital camera Olympus C7070 Wide Zoom• Image J program (Version 1.33). Methods1. Determination of equal PCR amplification efficiency of target and competitor sequences.a. Ensemble ten identical PCR tubes containing equimolecular quantities of target and competitor sequences in 50 µl as final volume. Use 0.2 µM of each specific primer and 25 µl of PCR Master Mix. Perform 3 min to 95°C to denature all DNA and a cycling program 30 s to 94°C, 30 s to annealing temperature according the Tm of the specific oligonucleotides, and 1 min to 72°C. Remove one tube at 17, 19, 21, 24, 27, 30, 33, 36, 39, and 45 cycles, respectively, and store at −20°C.b. Electrophoresis in 2% agarose gel in TA 1× (0.04 M Tris–acetate, 0.001 M EDTA, pH 7) of the amplification products of each tube.c. Take the digital images of the gel.d. Densitometry analysis of competitor and target bands in each lane using Image J Program (Version 1.33). This program is ideal to compare bands in the same digital image. It is based on the fact that optical density (OD) is a logarithmic function of brightness. A set of macros are bundled to Image J which are used for gel densitometry analysis producing curves. The height of the curve, at any given point, is the mean of the OD of a given row of pixels in the marked lane. The program can calculate area of user-defined selections. The area measurements are recorded in tabular form and are displayed in a Results window as shown in Fig. 7. To get more information, visit http://rsb.info.nih.gov/ij/e. Plot the area measurements versus cycle number to target and competitor, respectively (Fig. 3).f. Calculate efficiency to each cycle to target and competitor as was described in Section 2.g. Compare the efficiencies of target and competitor using a paired t test.h. According to the results, establish 30 cycles for all competitive PCRs.2. Determination of the sensibility of this method.a. Ensemble PCRs in the same conditions established before using decreased quantities of target with different dilutions of competitor with replicas for each point. In our experiment, we ensemble:• 2,000 molecules of target with 750, 1,000, and 5,000 molecules of competitor, respectively.• 1,000 molecules of target with 500, 1,000, and 5,000 molecules of competitor, respectively.• 750 molecules of target with, 250, 750, and 1,000 molecules of competitor, respectively.• 100 molecules of target with 50, 100, and 500 molecules of competitor, respectively.• 10 molecules of target with 5, 10, and 50 molecules of competitor, respectively.b. Electrophoresis in 2% agarose gel in TA 1× (0.04 M Tris–acetate, 0.001 M EDTA, pH 7) of the amplification products of each tube.c. Determination of minimum quantity of target that the method is able to detect. |
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