Profile MicroRNAs from Cancer and Normal Tissues

Numerous miRNAs has been found to have links with some types of cancer. Expression profiling of microRNAs can assist in identifying the type and severity of the oncogenic state and microRNA expression "signatures" can be used to develop diagnostics and therapeutics.

Measure both siRNA and mRNA knockdown in a single QuantiMir cDNA sample

Quantitate siRNAs and mRNA knockdown using QuantiMir: The use of sh/siRNAs to lower the levels of endogenous messenger RNA expression is a powerful and effective tool to study the roles of a targeted transcript in biological systems. Real-time qPCR has become the gold standard in accurately measuring the knockdown effects of specific sh/siRNAs in these experiments. The means of assessing the expression levels of sh/siRNA input has been typically achieved through laborious and nonquantitative Northern blot analyses. Here we present a new method to quantitate both the expression levels of sh/siRNA and the knockdown effect on the targeted messenger RNA transcript using a passive lysis buffer coupled with the QuantiMir kit cDNA synthesis and Real-time qPCR.

After transfection of siRNA or shRNA expression constructs, the cells are removed from the transfection plate using standard trypsinization. The cells are pelleted and subsequently lysed using 100 ul of SBI’s Cells-to-Cts passive lysis Buffer system. A 5 ul aliquot of the lysed cell suspension is then input directly into the QuantiMir RT reaction. The unique technologies in the QuantiMir RT kit enables the conversion of small RNA into detectable cDNA while simultaneously converting messenger RNA into cDNA. Accurate measurement of sh/siRNAs and the messenger RNA transcript in knockdown experiments can be accomplished using the cDNA created from this single RT reaction. The QuantiMir RT reaction generates enough cDNA suitable for numerous qPCR reactions. Shown below is an example of a timecourse study using anti-p53 shRNA-directed knockdown of the endogenous p53 mRNA transcript. From the same QuantiMir cDNA, both the p53 siRNA (Orange bars) and the p53 mRNA transcript (Blue line) were measured by qPCR.