UM_TotalRNA_NucleoZOL(18)

user's manuals about RNA or or

6.2 Troubleshooting

Problem

Possible cause and suggestions

Homogenization or sample lysis is incomplete

Insufficient yield

Improve homogenization by testing more stringent conditions.

Increase volume of RNase-free water for dissolving of RNA and prolong mixing for solubilization. Increase volume of NucleoZOL.

Use Buffer AE (5 mM Tris pH 8.5) for sample dilution for spectrophotometric quantification.

Increase volume of RNase-free water for dissolving of RNA and prolong mixing for solubilization.

Perform phase separation as described in section 5.2.3.Make sure to use fresh tissue or flash-freeze tissue immediately upon harvest.Store samples at -70 °C.

Make sure cells stay intact during trypsinization.Make sure to work in an RNase-free environment.

Solubilization of the RNA pellet is incomplete.

Volume of NucleoZOL used for homogenization was too low

Low pH during spectrophotometric quantification

Ratio A260/280 < 1.6

RNA pellet was only partly solubilized

Contamination of polysaccharide or proteoglycan

Inadequate tissue sampling

Inappropriate storage conditions

Degraded RNA

Cell dissolution during trypsinizationRNase contaminated solutions or tubes