Arabidopsis derived promoters for regulation of plant expression
a technology of plant genes and promoters, applied in the field of plant molecular biology, to achieve the effect of less biased analysis
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example 1
[0368]
GENECHIP ® Standard ProtocolQuantitation of Total RNATotal RNA from plant tissue is extracted and quantified.1. Quantify total RNA using GeneQuant 10D260 = 40 mg RNA / ml; A260 / A280 = 1.9 to about 2.12. Run gel to check the integrity and purity of the extracted RNASynthesis of Double-Stranded cDNAGibco / BRL SuperScript Choice System for cDNA Synthesis (Cat#1B090-019) was employed toprepare cDNAs. T7-(dT)24 oligonucleotides were prepared and purified by HPLC. (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGA GGCGG-(dT)24-3′; SEQ ID NO: 27).Step 1. Primer Hybridization:Incubate at 70° C. for 10 minutesQuick spin and put on ice brieflyStep 2. Temperature Adjustment:Incubate at 42° C. for 2 minutesStep 3. First Strand Synthesis:DEPC-water-1 μlRNA (10 μg final)-10 μlT7 = (dT)24 Primer (100 pmol final)-1 μl pmol5 × 1st strand cDNA buffer-4 μl0.1 M DTT (10 mM final)-2 μl10 mM dNTP mix (500 μM final)-1 μlSuperscript II RT 200 U / μl-1 μlTotal of 20 μlMix wellIncubate at 42° C. for 1 hourStep 4. Secon...
example 2
Characterization of Gene Expression Profiles During Plant Development Using the GENECHIP®
[0369]The Arabidopsis GENECHIP® provides a method to simultaneously scan over 30% of the genome for the expression profile of each gene on chip. By using RNA extracted from different tissue and developmental stages of development, a scan of the entire Arabidopsis plant is achieved. The advantages of a gene chip in such an analysis include a global gene expression analysis, quantitative results, a highly reproducible system, and a higher sensitivity than Northern blot analyses. Moreover, a gene chip with Arabidopsis DNA has a further advantage in that the Arabidopsis genome is well characterized.
[0370]Using the recently designed Arabidopsis high density oligonucleotide probe array, a total of 8,100 Arabidopsis thaliana genes were surveyed for temporal and developmental expression profiling. The objective was to identify known and novel genes that are expressed in specific organs (spatial expressi...
example 3
Further Analysis of Constitutively Expressed Genes
[0429]A standard curve of 50, 10, 2, 0.4, and 0.08 ng total RNA was generated for each primer / probe set tested. In this case, the 50 ng sample yielded a Ct value of 24.5 and the ng sample yields a Ct value of 26.7. The Ct value is defined as the threshold cycle whereby amplification occurs at an exponential rate. A low Ct value correlates with high gene expression. The threshold is determined empirically from the standard curve. By raising or lowering the threshold, the data set is maximized to represent optimal exponential amplification. A correlation coefficient (R2 of the best-fit line from the standard curve) greater than 99% and a slope of −3.3 (most efficient amplification) is ideal. For accurate repeatable results, the previous criteria must be met and the unknowns must fall within the range of the curve. The expression levels of the unknown can be interpolated from the unknown Ct values using the standard curve.
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