Figure 2. Assembly for freezing six microtiter plates of worms. Cut the foam rubber from a New England Biolabs Styrofoam container as directed and assemble the foam rubber pieces, six microtiter plates and single tube of test frozen worms as shown. It is critical that the pieces fit together tightly to ensure proper freezing of the worm suspensions.
For each plate to be frozen, label a new freezing plate: a 96-well Falcon 3911 “Microtest III flexible assay plate”. These special, soft plastic plates will allow you to cut out individual wells with a razor blade so that an individual microtiter well can be thawed without having to thaw (and throw away) an entire plate. Label each plate twice to ensure that at least one label will be legible - these plates must be identified correctly possibly years later. There are special sticky tags (e.g., “sidewall tough-tags”) that are easy to write on that can be put on the sides of plates. These can come off in the freezer, so if you use these only do so for one of the two labels per plate.
In analytics, surface-enhanced Raman scattering (SERS) became a powerful tool to gain information from biomolecules. By the use of noble metal nanoparticles as SERS substrates, high enhancement factors can be achieved. On this poster, we present SERS spectra of red blood cells and their components: hemoglobin and cell membranes. We report SERS spectra of Hb using silver nanoparticles at very small nanoparticle : Hb molecule ratios, that is, under conditions relevant for SERS-based nanotoxicity experiments with red blood cells at high sensitivity. We show that the structural information obtained in the experiment is highly dependent on the conditions under which the interaction of nanoparticles with Hb molecules takes place. An excitation wavelength in the near-infrared region (785 nm) is used to prevent cell damage. The SERS spectra from isolated hemoglobin and cell membranes enabled us to assign the spectral information from whole red blood cells. In addition to NIR-excitation, Raman scattering of isolated oxygenated hemoglobin was also excited at shorter wavelengths, and yielded different enhanced signals of porphyrin and globin due to pre-/resonant enhancement. In contrast to previous work, where information about the hemoglobin protein structure is mainly obtained in resonant Raman experiments in the UV-range , in SERS we find globin bands as well as additional vibrations of the porphyrin, also at longer wavelengths. In addition to the information on hemoglobin structure, our results have important implications for our understanding of the interaction of red blood cells with nanoparticles .
We acknowledge funding by ERC starting grant No. 259432 (MULTIBIOPHOT) and the Australian Academy of science – scientific visits to Europe program.
Prepare dsRNA corresponding to gene of interest by transcription. Inject dsRNA into young adult hermaphrodites, wait for RNAi to take effect, then score progeny of injected mother. This is a modification of the protocol in .
Semiconductor quantum dots (QDs) are considered as a lately improved sector of nanomaterial with specific photophysical characteristics that increase the interest and potentiality of emerging what is known as fluorescent biosensors. For biosensing QD, they are advantageous over many of the conventional used protein-based fluorophores and organic dye that suffer from various chemical and photophysical liabilities. They include pH dependent, susceptibility to photo-bleaching, self-quenching at high concentrations, narrow absorption windows coupled to broad red-tailed emission spectra via small Stokes shifts, short-term aqueous stability and short excited state fluorescent lifetimes involve broad absorption spectra coupled to narrow size tunable photo fluorescent emissions. On the other hand QDs are characterized by having high quantum yields and unique resistance for both chemical degradation and photobleaching. The current research studies the synthesize of the CdSe core with a layer of wider band gap semiconductor such as CdTe QDs with different sizes for investigating their efficiency and sensitivity in adapting QDs for biosensing applications as generalized probes, in glycoproteins detection using laser induced florescence and fluorescence resonance energy transfer (FRET) - based sensing. The study as well focused on the selection of materials and the methodology for attaching biomolecules to the QDs that relies on direct interaction with the QD surface through dative thiol bonding of cysteine residues to the surface sulfurs.
Grow desired worm strain on standard NGM plates seeded with OP50 bacteria. Carry out standard bleaching/washing protocol to obtain embryos, and leave to hatch into L1s overnight in M9 buffer. These starved L1s will be synchronized at the beginning of the L1 stage. If feeding will be done with larvae older than L1, then put hatched L1s onto standard NGM plates containing OP50 and grow to the desired stage.
RIVERSIDE, Calif. — Transcription is a cellular process by which genetic information from DNA is copied to for protein production. But anticancer drugs and environmental chemicals can sometimes interrupt this flow of genetic information by causing modifications in DNA.
Synopsis: Grow RNAi bacteria and seed plates. Feed worms with RNAi bacteria and score for phenotypes. The following is based on the protocol in .
Chemists at the University of California, Riverside have now developed a test in the lab to examine how such DNA modifications lead to aberrant transcription and ultimately a disruption in protein synthesis.
More than one gene can be targeted simultaneously by the soaking method in wild-type animals. Because the efficiency of double RNAi might be lower than that of single RNAi for each gene, protein or mRNA levels for each gene should be assessed whenever possible.
This part of the protocol will differ slightly depending on your assay. After feeding, either the aliquoted worms or their progeny can be scored. For some assays, scoring is easier if progeny are synchronized. In this case, fed gravid mothers are allowed to lay eggs on a new plate, then removed, and the progeny subsequently scored. This step is time consuming and not always necessary.
Typically, 24 hours post-injection is a good starting point for a good RNAi effect. For many genes, the strength and penetrance of RNAi phenotypes are increased in progeny laid more than 24 hours post-injection, especially for genes with a strong maternal contribution. It is a good idea to do a time course to find the optimum time of scoring post-injection, looking for a time when the phenotype is strongest and most penetrant. For some genes, shorter times post-injection will give a stronger effect, particularly for genes with a zygotic but not a maternal function. If antibodies are available, it can be helpful to stain progeny at different times post-injection to see when the protein is maximally reduced. In order to maintain progeny production at later time points, mate the injected hermaphrodites with N2 males after injection. Using an RNAi supersensitive strain can increase the strength and penetrance of phenotypes. and both display sterility at 25°C and smaller broods than N2 at lower temperatures (; ), but this can be overcome by mating with N2 males after injection, as they are cross fertile at all temperatures (J. Ahringer, unpublished).
“Aberrant transcription induced by DNA modifications has been proposed as one of the principal inducers of cancer and many other human diseases,” said , a professor of , whose lab led the research. “CTAB can help us quantitatively determine how a DNA modification diminishes the rate and fidelity of transcription in cells. These are useful to know because they affect how accurately protein is synthesized. In other words, CTAB allows us to assess how DNA damage ultimately impedes protein synthesis, how it induces mutant proteins. ”
When targeting more than one gene by RNAi, the efficiency of knockdown may be reduced when compared to targeting each gene separately (e.g., see Table 1 in ). This may not be a problem for some experiments. For example, if two genes each have no detectable RNAi phenotype, but targeting them together causes a phenotype, then this would show some overlap in function. However, if you are carrying out an epistasis experiment, reduced RNAi efficiency may lead to an inaccurate result, if only one of the two genes is efficiently knocked down. Therefore, it is ideal to target one gene by RNAi in the mutant background of the other gene. If it is not possible to use a mutant, then use antibodies to test that the level of protein knockdown is the same in the double and single RNAi experiments for both genes. Use of or RNAi supersensitive strains (; ; Wang et al., 2005) enhances the effectiveness of targeting multiple genes (S. Woods, D. Rivers, and J. Ahringer, unpublished; A. Fraser, pers. comm.).