Comparative analysis of rodent tissue preservation methods and nucleic acid extraction techniques for virus screening purposes

The polymerase chain reaction (PCR) has become an essential method for the detection of viruses in tissue specimens. However, it is well known that the presence of PCR inhibitors in tissue samples may cause false-negative results. Hence the identification of PCR inhibitors and evaluation and optimization of nucleic acid extraction and preservation methods is of prime concern in virus discovery programs dealing with animal tissues. Accordingly, to monitor and remove inhibitors we have performed comparative analyses of two commonly used tissue storage methods and five RNA purification techniques using a variety of animal tissues, containing quantified levels of added MS2 bacteriophages as the indicator of inhibition.The results showed (i) no significant difference between the two methods of sample preservation, viz. direct storage at −80 °C or 4 °C in RNAlater, (ii) lung rodent tissues contained lower levels of inhibitor than liver, kidney and spleen, (iii) RNA extraction using the EZ1 + PK RNA kit was the most effective procedure for removal of RT-PCR inhibitors.

► We investigated PCR inhibition depending on the pretreatment of rodent tissue samples. ► The degree on PCR inhibition was equal when using storage in RNAlater or at −80 °C. ► Pretreatment with proteinase K allowed reducing considerably the degree of inhibition. ► Extraction using EZ1 or RNAnow allowed a better purification as regarding inhibitors. ► The degree of inhibition was the highest in liver and the lowest in lung tissue.