The Use of the SV Total RNA Isolation System - Lab Report Example

Running head: DNA Lab Report DNA Lab Report [Writer’s Name] [Institution’s Name] Abstract The purity and integrity of RNA isolated from tissue or cultured cells are critical for its successful use in applications such as reverse transcription PCR (RT-PCR), real-time PCR, RNase protection assays, Northern blot analysis, oligo (dT) selection of poly (A) + RNA, in vitro translation and micro array analysis. In the current years, a powerful technique that is used to recognize and measure quantitate definite mRNAs from little amounts of total RNA and mRNA, RT-PCR has come out. Te requirement for techniques to segregate high-quality RNA swiftly, free of genomic DNA infectivity significantly, from small amounts of starting material (i.e., tissue and cultured cells) has grown additionally as the use of amplification as a research tool has developed. The solution for such requirements is the SV Total RNA Isolation System. For the preparation of disinfected and unharmed total RNA from tissues, refined cells and white blood cells in even an hour span, depending on the number of samples to be processed, a quick and uncomplicated technique is endowed with by the SV Total RNA Isolation System. Relying upon the kind, purpose and RNA appearance levels of the tissue, near about 60mg of tissue can be dealt with in a single decontamination. To trim down genomic DNA to a large extent, which can impede with the amplification-based techniques, a DNase treatment step has been added by the system as well. Without using phenol: chloroform extractions or ethanol precipitations, decontamination can be attained and no existence of DNase postpones are there in the final RNA preparation. So, the use of the SV Total RNA Isolation System makes it far easier to obtain a contamination proof RNA to be used in further efficient way to be used in further experimentation. Table of Contents 1. Introduction............................................................................................................................ a. Plant Transformation b. Green Fluorescent Protein (GFP) c. Transgene Expression d. Detecting Transgenes and their Expression e. Statement of the Purpose f. Statement of the Findings g. Definition of Terms 2. Methods.................................................................................................................................. a. Day 1 i. PCR detection of GFP transgene in transgenic Arabidopsis using intact leaf tissue as template DNA Extraction PCR Solution ii. Extraction of RNA for measurement of GFP levels in transgenic Arabidopsis by quantitative real time RT-PCR iii. Transient expression of GFP in Nicotiana benthamiana using Agrobacterium tumefaciens iv. Stable transformation of Arabidopsis by the floral dip method b. Day 2 i. Agarose gel electrophoresis of PCR products ii. Preparation of cDNA template for quantitative real-time PCR c. Day 3 3. Results.................................................................................................................................... a. 214 Transformed b. 218 Silenced c. Dissociation Curve d. Delta Rn vs. Cycle e. Calculation i. To compare between transformed and actin ii. To compare the silencing 4. Discussion.............................................................................................................................. List of Figures 1. Figure 1. Tobacco 214 2. Figure 2. Tobacco 214 + p19 3. Figure 3. Arabidopsis 214 4. Figure 4. Tobacco 218 5. Figure 5. Tobacco 218 + p19 6. Figure 6. Arabidopsis 218 7. Figure 7. Dissociation Curve 8. Figure 8. Delta Rn vs. Cycle DNA Lab Report Introduction Plant Transformation The development of gene transfer technology for plant transformation had first taken place around two decades back. For the purpose of attaining a stable transformation of plant species the requirement is that there is an inoculation of plant tissue cultures using an Agrobacterium tumefaciens strain that has a recombinant Ti plasmid. Another way is through the bombardment of the tissue with the smaller recombinant E. coli plasmids that have one or two genes. It is very common to see cell transformation and plant regeneration in several of the dicotyledonous plant species. In this what happens is the insertion of the outside DNA into the plant cells/tissues. Such an insertion takes place using Ti plasmid of A. tumefaciens. The most suitable of the dicot species in this case of Agrobacterium-mediated transformation are the solanaceous species tobacco1, potato and tomato2. Other than just the stable transformation, one can also attain transitory indication of transgenes in plant tissues. This is possible through the infiltration of the tissue using A. tumefaciens that has a recombinant Ti plasmid. Another way is by the bombardment of tissue using the smaller recombinant E. coli plasmids. One can obtain a very high level of transient transgene expression in leaf tissue of Nicotiana benthamiana when infiltration is done of it with A. Tumefaciens3 4. Green Fluorescent Protein (GFP) The green fluorescent protein (GFP) is a protein made up of 238 amino acids. When blue light is shown on it, it displays bright green fluorescence5. GFP gene is made use of in cell and molecular biology. In this field the use of this gene is as a reporter of expression6. With a little modification, GFP has been used for making biosensors7. Several animals have also been formed through which there is the proof-of-concept of GFP that there can be the expression of a gene in the whole organism. The introduction of GFP gene can be done in organisms and their maintenance follows in their genome. This maintenance is by means of breeding, injection using a viral vector, or through cell transformation. There has been the introduction and expression of the GFP gene in several bacteria, yeast and different fungi, fish, plant, and mammalian cells, which also includes human. Transgene Expression Transgene expression in plants is said to be very uneven8. The determination of this is the copy number of the transgene and also the site of the transgene within the genome. Apart from this being so variable, gene silencing can lead to the total inactivation of the expression. The silencing of the transgenes can take place at two levels: the transcriptional or post-transcriptional9. Detecting Transgenes and their Expression The detection of transgenes can be done through DNA-based methods, RNA-based methods, protein-based methods, linked reporter genes (such as GFP), biochemical assays and directly detecting a phenotype conferred by the transgene. DNA-based methods consist of PCR and Southern hybridization analysis. PCR is said to be a quick and sensitive procedure. However, the limitation in this method is the contamination of plasmid DNA. On the other hand, Southern hybridization happens to be not so fast and is also difficult. However, this is more reliable but only if the design of the experiment is good enough. Statement of the Purpose Stable transformation of Arabidopsis for GFP expression. Definition of Terms Plant transformation – “the process by which DNA is introduced into plant cells or tissues”10. Polymerase Chain Reaction (PCR) – the process through which duplication is done of “millions of identical copies of individual genes from a tiny sample”11. Ti plasmid – “of Agrobacterium are large circular DNA molecules”12. Methods DAY 1 PCR detection of GFP transgene in transgenic Arabidopsis using intact leaf tissue as template DNA Extraction Three kinds of grounded leaf were used in this case. There was WT, pUQC214 and pUQC218. First of all leaf tissue was sampled and this was done by pinching a disc from one leaf. The tissue was then grounded using a pestle in the tube. The WT grounded leaf was added to 400 μL of extraction buffer. The mixture was vortexed for about 5 seconds after which the extracts were centrifuged at 13,000 rpm for a minute. After this 300 μL of the supernatant was transferred to a new/fresh Eppendorf tube. This was done twice and so there were two tubes of this mixture. The above step was also carried out for pUQC214 grounded leaf and pUQC218 grounded leaf. Thus, in total there were six tubes collected, two each of WT, pUQC214 and pUQC218 grounded leaf. In another tube 400 μL of extraction buffer was poured. After this a fresh tube was taken. In this tube was poured 300 μL of the supernatant from the WT leaf and 300 μL of isopropanol. This solution was mixed gently after which it was made to stand for 2 minutes at room temperature. Following this centrifuging of the mixture was done for 5 minutes at 13,000 rpm. This step was carried out for all the supernatants (WT, pUQC214 and pUQC218) collected and again there were six test tubes. In the seventh test tube the 400 μL of extraction buffer and 300 μL of isopropanol was poured. This solution was treated in the same way; that is, it was centrifuged for 5 minutes at 13,000 rpm. After this the supernatant was discarded and the Eppendorf test tubes were allowed to blot on a clean tissue. The pellets were then made to dry for 15 minutes. In the next step the DNA pellets were re-suspended in H2O. PCR Solution After this the PCR solution was made. The solutions used for this are given in the following table: Stock* Volume (μL) 10x PCR buffer* 15 10x dNTPs* 15 Taq polymerase (5 units per μL) 1.5 10 μM GFP-BamHI 15 10 μM GFP-330 15 10 μM ERF-A 15 10 μM ERF-B 15 Sterile distilled water (SDW) 58.5 *10x PCR buffer = 500mM KCI, 15mM MgCl2, 1 M Tris pH 8); 10x dNTPs (i.e. 2.5 mM of each deoxy-nucleotide: dATP, dGTP, dTTP and dCTP) 7 test tubes were labelled. Following the labelling 19 μL of the PCR solution was dispensed into the 7 labelled test tubes. These tubes were on ice. 1 μL of the DNA template from the first test tubes was added to the corresponding new test tubes. The PCR machine was preheated to 85°C after which the tubes were loaded into that heated machine. The programming of the PCR machine had been done according to the following specifications: 10 cycles of: 15 secs @ 94°C 15 secs @ 72°C 25 cycles of: 15 secs @ 94°C 15 secs @ 55°C 15 secs @ 72°C Extraction of RNA for measurement of GFP levels in transgenic Arabidopsis by quantitative real time RT-PCR The sample was taken from pUQC214 in a test tube. In the tube was added 175 μL SV RNA buffer. Following this 350 μL of SV RNA Dilution Buffer (blue) was added to the tube. The contents of the tube were mixed and this was done by inverting for 3-4 minutes. The mixture was then heated at 70°C for 3 minutes. Following this was centrifuging done of the tube. This was done for 10 minutes. The cleared lysate was shifted to another test tube. To this new tube was added 200 μL of 100% ethanol and the contents were mixed properly. This mixture was then transferred to a spin basket assembly and it was centrifuged for a minute. The eluate was discarded. Following this 600 μL of SV RNA wash solution (along with ethanol) was added to the test tube which was then centrifuged for a minute. The eluate was again discarded. The next step was the preparation of the DNase incubation mix. The solutions for this are given in the following table: Solution Volume Yellow Core Buffer 40 μL MnCl2, 0.09M 5 μL DNase 1 5 μL These solutions were mixed gently. The next step was the application of 50 μL of DNase to membrane. This was then incubated at room temperature for 15 minutes. 200 μL of SV DNase Stop Solution, along with ethanol, was then added to the tube and centrifuged for 1 minute. 600 μL of SV RNA Wash Solution was added and centrifuged for a minute. It was then emptied. Then 250 μL of SV RNA Wash Solution was added. Centrifugation was done for 2 minutes and then it was emptied. The last solution added was 100 μL of Nuclease-Free Water to membrance and centrifuged for a minute in order to elute the RNA. It was then stored at -70°C. Transient expression of GFP in Nicotiana benthamiana using Agrobacterium tumefaciens The wild type of Nicotiana was treated with pUQC214. The syringed was pressed on the underside of the leaf and it was left for a week. Stable transformation of Arabidopsis by the floral dip method The flower of the Arabidopsis was submerged and then mildly agitated for 3-5 seconds. The transformed plant is then grown normally. DAY 2 Agarose gel electrophoresis of PCR products 2g of agarose was added to 100ml of 1xTBE buffer. We did this for two groups in order to save time. Agarose was melted in a microwave oven. 10 μL of SYBR SafeTM gel stain was added to the agarose after it had cooled down. Then 4 μL of 10xgel loading buffer was added to each of the tubes. This was then loaded into the wells in the gel (10 μL). The determination of the size of the PCR products was done through 1kb size marker (10 μg of DNA). It was then electrophoresed at 100V till the bromophenol blue was near the bottom of the gel. The gel photographed under UV light. Preparation of cDNA template for quantitative real-time PCR There were two tubes taken. These were labelled as A and B. In these tubes was poured 5.5 μL RNA solution and 0.5 μL (50 μM random hexanes). It was then mixed and incubated at 70°C for 10 minutes and then it was stored on ice. This results in single stranded cDNA template and primers. Following this was added RT buffer, DTT and deoxyribonucleoside triphosphates. The proportion in which this was added is given in the following table: Stock Volume 5 x RT buffer 2 μL 0.1M DTT 1 μL 10mM dNTPs 0.5 μL This solution was then mixed and incubated at 42°C for 5 minutes. In the test tube A reverse transcriptase was added – 0.5 μL Superscript II RNaseH minus Reverse Transcriptase. This treatment was not given to test tube B as it was the negative control. Both the test tubes were then incubated at 42°C for 50 minutes (RT reaction). After this it was incubated at 70°C for 15 minutes. After this the reactions were diluted to 50 μL using sterile distilled water and then it was stored on ice till it was used as template in quantitative real-time PCR. DAY 3 Our group position in the PCR is B3. However, due to the fact that the actin cycle for this sample was more than 30 (32.05 to be exact) which is not effective, we chose another sample (sample 8) for transformation expression and (sample 17) for effective silencing. Results 214 Transformed Figure 1. Tobacco 214 Figure 2. Tobacco 214 + p19 Figure 3. Arabidopsis 214 218 Silenced Figure 4. Tobacco 218 Figure 5. Tobacco 218 + p19 Figure 6. Arabidopsis 218 The PCR result was not accurate and that may be because the primer was quite old. Figure 7. Dissociation Curve Our results were accurate and acceptable. Actin had lower break temperature when compared with GFP. Actin was used as control; it has a shorter base when compared with our GFP target. Therefore, actin had a lower temperature than GFP. Figure 8. Delta Rn vs. Cycle Our experiment result was acceptable because our data was lower than 30 cycle time. The calculation Samples used were 8 and 17. (214) control = transformed (218) sample = silenced To compare between transformed and actin: ΔCT = CT sample (214) – CT references (actin) = [(21.02 + 21.05)/5] – [(28.32 + 28.52)/2] = 21.035 – 28.42 ΔCT = -7.39 ABI expression – [target gene/reference gene] = 2-ΔCT = 2-(-7.39) = 167.73 So, GFP expression in the transformed 214 is 167.73 times more than the actin expression in the transformed plant. To compare the silencing ΔΔCT = ΔCT sample (218) – ΔCT control (214) ΔCT Sample = [(24.84 + 25.21)/2] – [(25.18 + 25.36)/2] = 25.03 – 25.27 = -0.24 ΔCT Control = [(21.02 + 21.05)/2] – [(28.32 + 28.52)/2] = 21.035 – 28.42 = -7.385 ΔΔCT = -0.24 – (-7.385) = -0.24 + 7.385 = 7.145 ABI expression - [target gene/reference gene] = 2-ΔΔCT = 2-(7.145) = 7.065 X 10-3 (7.065 X 10-3) X 100 = 0.7% 100% - 0.7% = 99.3% The silencing is 99.3% effective. References Chawla, H. S. Introduction to plant biotechnology, Science Publishers, New Hampshire, 2002. Fillatti, J.J., Kiser, J., Rose, R., & Comai, L. ‘Efficient transfer of a glyphosate tolerance gene into tomato using a binary’, Agrobacterium tumefaciens vector. Bio/Technology Vol.5, 1987, pp.726-730. Grace, E. S. & National Academies Press (U.S.). Biotechnology unzipped: promises and realities, Joseph Henry Press, Washington, DC, 2006. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G., & Fraley, R.T. A simple and general method for transferring genes into plants. Science, Vol. 227, issue 4691, 1985, pp.1229–1231. Elsas, J. D., Jansson, J. K., & Trevors, J. T. Modern soil microbiology, CRC Press, Boca Raton, FL, 2007. Mallory, A.C., Ely, L., Smith, T.H., Marathe, R., Anandalakshmi, R., Fagard, M., Vaucheret, H., Pruss, G., Bowman, L., & Vance, V.B. ‘HC-Pro Suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal’, Plant Cell, Vol. 13, 2001, pp.571-583. Mallory, A.C., Reinhart, B.J., Bartel, D., Vance, V.B., & Bowman, L.H. 2002. ‘A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco’, Proc. Natl. Acad. Sci. USA, Vol. 99, 2002, pp.15228-15233. Matzke, M.A., Matzke, A. J. M., Pruss, G. J., & Vance, V. B. ‘RNA-based silencing strategies in plants’, Curr. Opin. Genet. Dev, Vol.11, 2001, pp.221-227. Meier, I. 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