Protector RNase Inhibitor Protocol & Troubleshooting

Product No. RNAINH-RO

Protocol

ADDITION OF PROTECTOR RNASE INHIBITOR TO CELL PELLETS

The addition of Protector RNase Inhibitor to cell pellets is not recommended. Protector RNase Inhibitor as a 50kDa protein will most probably not permeate into intact cells. If the cell pellet is immediately frozen at -80 °C, this will be sufficient for stabilizing RNA. At -80 °C, Protector RNase Inhibitor is inactive.

ADDITION OF PROTECTOR RNASE INHIBITOR PRIOR TO ISOLATION OF RNA

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

The intended application of Protector RNase Inhibitor is to be added to purified RNA and to protect RNA in reaction mixtures, such as RT-PCR and in vitro transcription reactions.

Troubleshooting

LOW RNA YIELD FROM PRIMARY MONONUCLEAR CELLS

It is not an option to add Protector Protease Inhibitor directly to primary mononuclear cells.
Please consider the following when purifying RNA from such cells:
Primary cells and immortalized culture cell lines are very different. Immortalized culture cells have a completely 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 probably have more ribosomes than primary cells. The major part of total RNA is ribosomal RNA which is consistent with a much greater RNA yield from culture cells. For these reasons, it may be helpful to switch to an mRNA isolation procedure instead of isolating total RNA.

It is the best not to have a long time span between harvesting the cells and performing the RNA isolation. When the incubation period with lipopolysaccharides is completed, cells should be immediately washed with ice cold PBS to keep metabolic activities to a minimum. Cell culture vials and plates should also be kept on ice until the cells are lysed using lysis buffer.

Materials

     
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