plz suggest the easiest overall total lipids method for small amount of tissue and its calculation .?
I have no experience with this myself, but I found this item that might be useful:
Source: http://www.cyberlipid.org/extract/total1.htm
Colorimetric methods are mainly used to estimate lipid content in very small samples (some mg) or in small aliquots of large samples.
A micro-colorimetric assay have been developed for a single mosquito, and should be reliable for other small tissue samples (E. Van Handel, J Am Mosq Control Assoc 1985, 1, 302).
Briefly, lipid samples and standards are placed in a heating block set at 100°C to allow the solvent to evaporate. Once the solvent is gone (about 10 min), 0.1 ml of concentrated sulfuric acid is added to each tube, vortexed, and then heated at 100°C for 10 min. Samples are then removed from the heat block and allowed to cool to room temperature before adding 2.4 ml of vanillin reagent (600 mg of vanillin, 100 ml of hot water, 400 ml of 85% phosphoric acid) and vortexed. The pink color is allowed to develop for 5 min, and then 0.2 ml of standards and samples are read at 490 nm. A standard spectrophotometer can be used, but a 96-well spectrophotometer decreases analysis time and increases accuracy by reading of standards at virtually the same time as the samples. Since the color continues to develop over time, the standards should be re-read at 5 min intervals if a standard spectophotometer is used.
An accurate adaptation of the micro-colorimetric method was described using quantification of lipids in a 96-well microplate enabling higher throughput and reduced costs (Cheng YS et al., Lipids 2001, 46, 95).
When compared with two other approaches, the reported procedure was shown to be the most reliable (Lu Y et al., Lu Y et al., Can J Fish Aquat Sci 2008, 65, 2233).
You will need to make a standard curve with several known quantities of lipid that give a signal in the dynamic range of the assay. Then you can compare the signal from your own samples to the standard curve to determine the amount of lipid in your samples. Then by calculating the proportion of the original material that was used in the assay, you can figure out how much lipid was in the original sample.
I have successfully used the dye in the link below to develop an assay to determine the total lipids in solution (see link below).
Make a calibration curve using a suitable standard (paying close attention to your sample matrix) on a fluorometer or using your fluorescent plate reader, and run your unknown against the standard curve.
http://www.setabiomedicals.com/cholesterol-determination.html
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