Protector RNase Inhibitor Protocol & Troubleshooting

Protector RNase Inhibitor (RNAINH-RO) inactivates a wide spectrum of RNases, including RNase A, RNase B, and RNase T2. This RNase inhibitor can help prevent RNase degradation in any application where RNases could interfere with desired results.

Inhibiting RNase Activity

Add Protector RNase Inhibitor to purified RNA and RNA reaction mixtures, such as RT-PCR and in vitro transcription reactions.

Cell Pellets: Protector RNase Inhibitor is a 50 kDa protein and is unlikely to permeate into intact cells. Therefore, adding Protector RNase Inhibitor to cell pellets is not recommended. Immediately freezing the cell pellet at -80 °C is sufficient for stabilizing RNA. Protector RNase Inhibitor is inactive at -80 °C.

Cell Lysates: The addition of Protector RNase Inhibitor to cell lysates before RNA isolation using High Pure RNA Isolation Kit is not recommended. The lysis buffer of the High Pure RNA Isolation Kit contains RNase inhibiting compounds and these protein denaturing compounds will inactivate Protector RNase Inhibitor.

Troubleshooting

Primary Mononuclear Cells: Do not add Protector Protease Inhibitor directly to primary mononuclear cells.

Note that primary cells and immortalized culture cell lines are very different. Immortalized culture cells have different metabolism, cell size, cell density, and much faster growth rate, resulting in a larger endogenous content of RNA. Because growth rate is linked to protein synthesis, culture cells will typically have more ribosomes than primary cells. Most of the total RNA is ribosomal RNA, which is consistent with a much greater RNA yield from culture cells. Therefore, it may be helpful to use an mRNA isolation procedure instead of isolating total RNA.

It is best to have a short time span between harvesting the cells and performing RNA isolation. When the incubation period with lipopolysaccharides is completed, immediately wash cells with ice-cold PBS to keep metabolic activities to a minimum. Keep cell culture vials and plates on ice until the cells are lysed with lysis buffer.