Actin Quantification

Unfortunately these and other existing quality control procedures do not answer aU problems. There remains a clear need for development of PCR reference materials that win provide information both on quality and quantity levels. For quality the reference materials should be host-specific and PCR primers, for positive control, may correspond to host specific house keeping genes e.g. b-actin. For quantitative analysis, fluorescence dyes in specific primers might be used in order to measure accurately the amount of DNA present. Such practices, and other as yet un-realized procedures, will be needed to achieve reliable results in the quantification of DNA analysis. 

In this approach, bisulfite-treated DNA is used as the template, and 50-100nM nnmethylated or methylated DNA-specific primers are nsed for PCR amplification in separate reactions. For quantification, a AAC.J, method is nsed and normalized with the C.J, (cycle threshold) for the 3-actin gene (43). Alternatively, a fragment of the target gene promoter will be amplified using primers designed from a CpG-free area as an internal control (see Note 3). To eliminate any primer dimer that will compromise the accuracy of the results, an additional step in the PCR cycles above... 

Shot-noise-limited performance can be conveniently recognized by noting how the S/N of a system changes with light level. The S/N should vary quadratically with intensity if it is shot-noise-limited. Once a measnrement is determined to be shot-noise-limited, the quantification of the noise amplitnde provides a simple, direct, and effective way to estimate the actinic flnx experienced by a sample during a measnrement. ... 

Quantification of the amount of toxin-ADP-ribosylated actin in the intact cell can be done by the method of differential ADP-ribosylation. However, pP]ADP-ribosylation of cell lysates prepared by standard procedures is critical because C2I can still work on ice. Thus, under the conditions of short term intoxication or analysis of the time course of intoxication, C2I continues to ADP-ribosylate actin during the preparation of the lysates. This results in an apparently higher amount of actin modified in the intact cell. To avoid this artefact, it is recommended that lysis of cells should be performed in the presence of p2p]NAD (as given in 11.2.3.2 ). 

Fig. 8 Left-. C2C12 cells were seeded on aligned and randomly oriented fibers and on a glass control. Confocal images taken at 30 min, 60 min, 2 h, 5 h, and 24 h after seeding are shown, with increasing time from top to bottom. Actin is stained green and nuclei are stained blue. Scale bar. 25 pm. Right Quantification of cell aspect ratio is shown for all time points. All data sets are significantly different from each other except those indicated by NS [52]...

This method is also suitable for simultaneous quantification of several mRNA species (O Donovan et al., 1991). Different lengths of oligomers are used to the different mRNAs and an internal reference such as /3-actin. It is necessary that these probes have the same specific activity (e.g., by kinasing) and the same hybridization rate and stability. 

Figure 9 RT-PCR analysis of cytokine mRNA expression in the spleens of DBA/2 mice immunized with casein/CKA. Total RNA was extracted from pooled spleens of S DHA/2 mice or CVE-administercd mice 3 weeks after immunized using RNAzol and reverse-transcribed, and the cDNA concentration was adjusted by co-amplification of two fold serially diluted cDNA and constant amounts of control plasmid pMCQ with [5-actin specific primers. Cytokine expression patterns of IL-12, IFNy, IL-6, and 11.-10 were determined by PCR using murine cytokine-specific primers. The PCR products were separated in 2 % agarose gel and visualized by ethidium bromide staining. Relative quantification of the amounts of the RT-PCR products was performed using a Computing Densitometer and NIH Image software,...

FIGURE 109 Comparison between three DNA quantification methods used in the assay of a PCR product (347 bp fragment of the actin gene) from left to right UV absorption, PicoGreen -dye method, and [Eu(L13 )3]/AO method (redrawn after Song et al., 2008b). 

Sotelo, C.G., Pineiro, C., Perez-Martin, R.I., and Gallardo, J.M. 2000. Analysis of fish and squid myofibrillar proteins by capillary sodium dodecyl sulfate gel electrophoresis Actin and myosin quantification. Ear. Food Res. Technol. 211, 443-448. 

A) Knockdown of RARa in NIH3T3 mouse fibroblasts was conducted using two different shRNAs, shl, and sh2, compared to control (Ctr). (a) Representative immunoblot. Actin is shown as loading control and (b) amounts of RARa in control and knockdown cells determined by densitometric quantification of immunoblots represented by the one shown in (a). Values are normalized for actin and expressed as multiples of control (none) values n = 3. (B) Rates of degradation of long-lived proteins in control and RAR(-) cells maintained in the presence or absence of serum for 12h. Values are expressed as percentage of proteolysis ... 

The illumination was essential to increase the mRNA levels for ELIP. As can be seen in fig. 8. the levels for all three mRNAs fluctuate during the day with a minimum at around 17 i. e. 10 hrs after the temperature shift. Although the curves are somewhat different from those reported (7) there is a clear fluctuation of mRNA levels during the day. In order to exclude mistakes in the quantification of mRNAs the filters were also hybridized as internal standard to an actin probe (19) which does not oscillate during the day. 

Fig. 2 Increasing content recovery by coupling luciferase-based assays with high-throughput biochemical readouts. The same cell line subjected to the luciferase-based assay protocol (HCT116 cells) is evaluated here for its reliability In reporting a biochemical readout (p53 expression) by dot blot analysis. Cells transfected with indicated expression construct and pathway reporters in a 96-well culture plates were lysed 48 h posttransfection. Following luciferase activity measurements (data not shown), protein from spent lysates was immobilized on nitrocellulose using a liquid handler and filtration manifold, (a) p53 and p-actin protein levels detected using protein-specific antibodies, infrared fluorescent dye-coupled secondary antibodies (with emissions at 680 and 800 nM), and the Li-COR imaging system. Columns of lysate corresponding to cells transfected with p53 DNA are boxed, (b) Quantification of the p53 to p-actin protein ratio...

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