Functional Genomic Analysis of C. elegans Using RNA Interference

Introduction Before talking about this important experiment, do you know what nematode is? Specifically, the experiment used nematode nematodes called nematodes. Caenorhabditis elegans nematode Caenorhabditis elegans is a small worm (like what you found on the ground), but there is a very special place in modern biochemistry: scientists mapped the whole genome. This sequence allows scientists to know the nature and location of all nematode genes. However, since biochemists do not fully understand the role of each gene, the purpose of this experiment is to find the function of each gene in the worm.

Caplen et al. (2001) first examined the interference of C. elegans using siRNA corresponding to Caenorhabditis elegans unc-22 and investigated whether siRNA can directly inhibit gene expression. They produce dsRNA molecules by mixing sense and antisense ssRNA oligomers. The integrity of the annealed RNA and the characteristics of the dsRNA were confirmed by gel electrophoresis. All mammalian cells used were grown in DMEM primary mouse embryonic fibroblasts (MEF) from wild type I 129 mouse embryos and grown to produce more homogeneous cell lines. They then used standard methods for cell culture and nucleic acid transfection. Analysis of gene expression was performed using a series of techniques. GFP expression was assessed in mammalian cells by fluorescence activated cell sorter. To assess cell viability, Caplen et al. (2001) performed an in vitro kinase assay and observed labeled proteins by xerogel autoradiography.

This project initially isolated yeast RNA from cell culture. RNA was extracted and purified in several steps to obtain a purified sample. Purification of the sample was confirmed by spectrophotometric analysis of the RNA sample and measurement of the A260 / A280 ratio (Table 2). After RNA purification step, reverse transcriptase polymerase chain reaction (RT - PCR) is used to convert RNA to cDNA. Transformation of RNA into C cDNA template using reverse transcriptase derived from viral function is amplified to generate DNA portions containing bait and predation sequences (Oligos, 2010) (Sequences 1 and 2) . The amplification primer is derived from an oligonucleotide derived from the yeast genome. These oligonucleotides (MP1) for predation are ligated to the vector (pGADT7).

In order to manipulate the viral RNA genome, scientists of molecular biology era have developed an efficient enzymatic method to generate complementary DNA (cDNA) copies of viral genomic RNA using retroviral encoding reverse transcriptase . The cDNA can be designed to have a "sticky" end and the molecule can then be cloned into E. coli where it can be manipulated by all existing methods. This may also include deletion, deletion or point mutation of the protein coding sequence, or even the introduction of entirely novel protein coding sequences. The modified cDNA can then be placed downstream of a promoter sequence suitable for DNA-dependent RNA polymerase and a novel molecular engineered viral RNA genome that is efficiently transcribed in an in vitro transcription reaction.