One of the best characteristics for the functional status of a certain cell is its gene expression pattern. Cells belonging to different tissues, cells in different developmental or metabolic stages, cells under the influence of specific compounds, or cells within a carcinogenic process differ by their gene expression patterns and thus by their mRNA pools. Currently, the most important technique for the accurate quantitation of gene expression is the fluorescent quantitative real-time RT-PCR (Muller et al., 2002a).
Reverse transcription (RT) followed by the polymerase chain reaction (PCR) is the technique of choice to analyze mRNA expression derived from various sources. Real-time RT-PCR is highly sensitive and allows quantification of rare transcripts and small changes in gene expression. It is easy to perform, provide the necessary accuracy, and produce reliable as well as rapid quantification results (Pfaffl, 2001). Many of the key proteins (i.e. cytokines and transcription factors) are found in such low abundance that real-time RT-PCR quantification of their mRNAs represents the only technique sensitive enough to measure their expression reliably in vivo, low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity, (Huggett et al., 2005a)
RNA cannot serve as a template for PCR, the first step in an RT-PCR assay is the reverse transcription of the RNA template into complimentary DNA and followed by its exponential amplification in a PCR reaction. Usually, this involves the use of dedicated RNA and DNA-dependent DNA polymerases, either in separate (‘two-enzyme/two-tube’) or in single (‘two-enzyme/one-tube’) reactions, as the use of dedicated enzymes with different proper...
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...rse transcription polymerase chain reaction assays." Journal of molecular endocrinology 25.2 (2000b): 169-193. (article given)
3. Huggett, J., et al. "Real-time RT-PCR normalisation; strategies and considerations." Genes and immunity 6.4 (2005a): 279-284.
4. Huggett, J., et al. "Real-time RT-PCR normalisation; strategies and considerations." Genes and immunity 6.4 (2005b): 279-284.
5. Muller, Patrick Y., et al. "Short technical report processing of gene expression data generated by quantitative Real-Time RT-PCR." Biotechniques 32.6 (2002a): 1372-1379.
6. Muller, Patrick Y., et al. "Short technical report processing of gene expression data generated by quantitative Real-Time RT-PCR." Biotechniques 32.6 (2002b): 1372-1379.
7. Pfaffl, Michael W. "A new mathematical model for relative quantification in real-time RT–PCR." Nucleic acids research 29.9 (2001): e45-e45.
completed, the tubes are stored at 4°C until analysis of the tubes. To alylize the PCR results with
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
2) To establish the expression patterns and regulations of the genes using available microarray data.
...ta from sequence with high accuracy. It mostly used in signal-transduction study and allele-expression study and it was relative expensive compared with siRNA sequencing2, 4.
Predicting gene function revolves around predicting protein localisation and defining conserved functional domains. These are both dependant on whether the target sequence is prokaryotic or eukaryotic in origin, as different signalling peptides, possibilities for localisation and useful domains exist for each classification. However, gene expression data has been overlooked as a method of functional analysis as analysis of either classification follows a similar method. Gene expression data is useful as it further narrows the ambiguity of protein function to specific cellular events.
Q1Aamp DNA mini kit, leading to the second period being the amplification of 16s rDNA consensus sequence by PCR, particularly using the primers RW01 and DG74. The unknown sample is then taken for gel electrophoresis to confirm and purify the amplified 16s rDNA fragment, done in the third period of the experiment. Once the running of the gel is completed, a cut of the 370bp PCR fragment is taken, and is put for purification of 16s rDNA fragment by the QIAquick gel elution kit, allowing the DNA to elute at the bottom of the microcentrifuge tube
This data is used in DESeq66, which is an R Bioconductor package, to calculate differentially expressed genes between HPC and PFC. DESeq provides various statistical tests for determining differentially expressed genes in gene expression data67 The inputs for DESeq are raw counts obtained from HTSeq. DESeq takes into account the total size of each library to perform calculations on fold change as well as significance based on p-value and adjusted p-value. The transcript biotypes were obtained from the Ensembl GTF annotation file (Mus musculus genome build NCBIM37). Using the annotation file, we identified 34,379 transcripts from HPC and 32,909 transcripts from PFC. Analysis of this dataset by blasting against the EMBL database containing 2,057 lncRNAs led to the identification of 1,982 lncRNAs from HPC and 1,936 lncRNAs from PFC (Fig 1, from Kadakkuzha et al., submitted to Genome
4.) Mainardi, Paola. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Sept. 2006. Web. 29 Nov. 2013. .
Holton, T. A., and M. W. Graham. "A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors." Nucleic acids research 19.5 (1991): 1156.
Real-time PCR or qPCR allows the on going process in a cell to be monitored. This results in quantifiable amounts using fluorescent probes. Now the amount of gene expression can be compared over time. Therefore up or down regulation of transcription can be followed, for now a comparative measure can be obtained.
The results partially agreed with the working hypothesis. The student sample genotype was indeterminable, most likely due to PCR errors that
It is used in many labs and only requires the DNA in question, primers that anneal to the beginning and end of the target genes, Thermus aquaticus, Taq DNA, a heat stable DNA polymerase and all four of the deoxyribonucleate triphosphates. There are three steps in the PCR reaction denaturation, hybridization and DNA synthesis. During these steps the DNA is separated or denatured into two strands, hybridized, where the two single strands are complimentary paired to the respective primers, and then the DNA is synthesized with Taq DNA. This is considered one cycle, and it can commonly take 50 cycles to amplify enough DNA to be used. When the PCR is completed a gel electrophoresis is run. The PCR product is put in a specially formed agarose gel that will allow electricity to flow around the gel and DNA and force the DNA to travel down the gel resulting in white bands depending on their electronegativity. When the DNA is transformed from plasmid into the yeast we use salmon sperm to protect the nucleus from becoming degraded and the plasmid lost. This increases the efficiency of the DNA because the sperm DNA will adhere to the yeast cell wall and allow the plasmid to bind to the
Adnan, Amna. DNA Sequencing: Method, Benefits and Applications. 14 July 2010. 17 March 2014 .
Grinde, Bjørn, and Grete Grindal Patil. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 31 Aug. 2009. Web. 11 Apr. 2014.
Transcriptomics- the study of gene expression (Sorek & Cossart, 2010; Stewart, Sharma, Bryant, Eppley, & DeLong, 2011; Z. Wang, Gerstein, & Snyder, 2009)