RNA was isolated from 10 mg rat brain tissue using the RNeasy Lipid Tissue Mini Kit (RNeasy Lipid), a standard silica-gel-membrane procedure (Std. silica), or a phenol/guanidine-based reagent (PhOH/Gdn), following supplier's instruction. [A] Formaldehyde agarose gel analysis shows high yields of RNA using the RNeasy Lipid Tissue Mini Kit. Using the RNeasy and other silica-based methods, small RNAs (such as 5.8S rRNA, 5S rRNA, and tRNAs) are selectively excluded. [B] Absorbance spectrum shows contaminants and phenol when using the phenol/guanidine-based reagent.
The convenient RNeasy Lipid Tissue protocol integrates QIAzol phenol/guanidine-based lysis with silica-membrane RNeasy RNA isolation for high yields of total RNA. The combination of organic extraction and chaotropic disruption contributes to the high lysis efficiency of QIAzol Lysis Reagent, enabling use of larger amounts of fatty tissues in the RNeasy procedure. RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase. Ethanol is added to the aqueous phase and the sample is applied to the RNeasy spin column.
Total RNA was isolated from 100 mg rat brain or adipose tissues using the RNeasy Lipid Tissue Mini Kit. Real-time, quantitative RT-PCR was carried out using QuantiTect Probe RT-PCR Kit with the indicated amounts of total RNA (in duplicate) and primers and probe specific for the c-jun or alpha-actin genes.