Evaluation of Different RNA Extraction Methods from Agropatch Suppressor Assay for Small Quantities of Plant Tissue and Their Application for Analysis of Gene Expression
Keywords:agro infiltration; q-PCR; RNA extraction; RNA silencing; small tissue samples; TRIzol; viral suppressors of RNA silencing
The agroinfiltration assay provides fast and efficient way to transiently express genes into plant cells by Agrobacterium tumefaciens. Extraction of RNA of high quality and sufficient amounts is prerequisite for gene expression studies such as quantitative Real Time PCR (q-PCR) from infiltrated areas in agropatch suppressor assay with small quantities of plant tissue. To attain prime RNA extraction from small tissues of infiltrated N. benthamiana plants with Potato virus A helper component proteinase viral suppressor protein, the efficiency of three RNA extraction methods (LiCl, TRIzol reagent and commercial kit) was evaluated. The total RNA yield with LiCl method was 2.83 and 33.2-fold greater than that of TRIzol reagent and commercial kit, respectively. Also, total RNA yield using TRIzol reagent was 11.7-fold higher than that with commercial kit. The A260/A280 ratio mean for TRI reagent (1.95) and kit (1.9) extractions were within the optimum range.q-PCR revealed that the cycle threshold values of housekeeping gene, EIF-1Î± and target genes AGO1 and ATG6 for RNA extracted using LiCl and kit were 1.07 to 1.3 and 1.02 to 1.12 times higher than those evaluated with the TRIzol method. Overall, TRIzol method showed the most effective approach for obtaining RNA from N. benthamiana patches in gene expression studies.
Alemzadeh A, Fujie M, Usami S, Yamada T (2005). Isolation of high-quality RNA from high-phenolic tissues of eelgrass (Zostera marina L.) by keeping temperature low. Plant Molecular Biology Reporter 23(4):421.
Ballut L, Drucker M, Pugnière M, Cambon F, Blanc S, Roquet F, et al., Badaoui S (2005). HC-Pro, a multifunctional protein encoded by a plant RNA virus, targets the 20S proteasome and affects its enzymatic activities. Journal of General Virology 86:2595-2603.
Brigneti G, Voinnet O, Wan-Xiang L, Ding SW, Baulcombe DC (1998). Viral pathogenicity determinants are suppressors of transgene silencing. The EMBO Journal 17(22):6739-6746.
Ding SW, Li H, Lu R, Li F, Li WX (2004). RNA silencing: a conserved antiviral immunity of plants and animals. Virus Research 102(1):109-115.
Ding SW, Voinnet O (2007). Antiviral immunity directed by small RNAs. Cell 130(3):413-426.
Duncan DB (1951). A significance test for differences between ranked treatments in an analysis of variance. Virginia Journal of Science 2:171-189.
Germundsson A, Savenkov EI, Ala-Poikela M, Valkonen JP (2007). VPg of Potato virus A alone does not suppress RNA silencing but affects virulence of a heterologous virus. Virus Genes 34(3):387-399.
Johansen LK, Carrington JC (2001). Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiology 126(3):930-938.
Kasschau KD, Carrington JC (1998). A counter defensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95(4):461-470.
Li F, Ding SW (2006). Virus counter defense: diverse strategies for evading the RNA-silencing immunity. Annual Review of Microbiology 60:503-531.
Loulakakis KA, Roubelakis-Angelakis KA, Kanellis AK (1996). Isolation of functional RNA from grapevine tissues poor in nucleic acid content. American Journal of Enology and Viticulture 47(2):181-185.
Maciel BM, Dias JC, Romano CC, Sriranganathan N, Brendel M, Rezende RP (2011). Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of q-PCR and bacteriological culture methods. Genetics and Molecular Research 10(4):2578-2588.
Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Kimura T (2007). Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. Journal of Bioscience and Bioengineering 104(1):34-41.
Pirone TP, Blanc S (1996). Helper-dependent vector transmission of plant viruses. Annual Review of Phytopathology 34(1):227-247.
Qu F, Morris TJ (2005). Suppressors of RNA silencing encoded by plant viruses and their role in viral infections. FEBS Letters 579(26):5958-5964.
Rajamäki ML, Mäki-Valkama T, Mäkinen K, Valkonen JPT (2004). Infection with potyviruses. In: Talbot NJ (Ed) Plant-Pathogen Interactions. Blackwell, Sheffield pp 68-91.
Redondo E, Krause-Sakate R, Yang SJ, Lot H, Le Gall O, Candresse T (2001). Lettuce mosaic virus (LMV) pathogenicity determinants in susceptible and tolerant lettuce varieties map to different regions of the viral genome. Molecular Plant-Microbe Interactions 14(6):804-810.
Rojas MR, Zerbini FM, Allison RF, Gilbertson RL, Lucas WJ (1997). Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237(2):283-295.
Roth BM, Pruss GJ, Vance VB (2004). Plant viral suppressors of RNA silencing. Virus Research 102(1):97-108.
Sáenz P, Salvador B, Simón-Mateo C, Kasschau KD, Carrington JC, GarcÃa JA (2002). Host-specific involvement of the HC protein in the long-distance movement of potyviruses. Journal of Virology 76(4):1922-1931.
Sahana N, Kaur H, Basavaraj TF, Jain RK, Palukaitis P, Canto T, Praveen S (2012). Inhibition of the host proteasome facilitates Papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HC-Pro. Plos One 7(12), e52546 10.1371.
Sambrook J, Russell D (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press (3rd ed), New York.
Schultz DJ, Craig R, Cox-Foster DL, Mumma RO, Medford JI (1994). RNA isolation from recalcitrant plant tissue. Plant Molecular Biology Reporter 12(4):310-316.
Urcuqui-Inchima S, Haenni AL,Bernardi F (2001). Potyvirus proteins: a wealth of functions. Virus Research 74(1):157-175.
Voinnet O (2005). Induction and suppression of RNA silencing: insights from viral infections. Nature Reviews Genetics 6(3):206.
Wang Y, Gaba V, Yang J, Palukaitis P, Gal-On A (2002). Characterization of synergy between Cucumber mosaic virus and potyviruses in cucurbit hosts. Phytopathology 92(1):51-58.
Winfrey MR, Rott MA, Wortman AT (1997). Unraveling DNA: Molecular Biology for the Laboratory. Prentice-Hall, Upper Saddle River, NJ.
Zhu B, Gao KS, Wang KJ, Ke CH, Huang HQ (2012). Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium. Chemosphere 87(4):397-403.
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