Troubleshooting of RNA isolation methods in Papanicolaou HPVinfected smears

Abstract

Cervical cancer is one of the leading causes of cancer in women, worldwide. Infection with human
papillomavirus (HPV) has been accepted as the primary cause for the development of invasive cervical
cancer and its precursor lesions. Despite HPV infection has been proposed as an indispensable factor for
cervical cancer development, only a subset of neoplastic lesions with HPV infection persist and progress to
invasive cancer. This suggests us that other molecular events are also involved in cancer progression. Aim
of this study was to extract mRNA from cytobrush-collected healthy and HPV infected cervical epithelial
cells and investigate various RNA extraction and purification protocols for assessment of RNA yield and
quality. Taking into consideration that cervical cancer screening is based on the cytology based
Papanicolaou test (Pap test), main challenge is to investigate whether the samples obtained by regular Pap
testing can be used for gene expression analysis. For this purpose, a total of 68 cervical specimens were
previously tested for HPV infection. Following HPV testing, samples were submitted to RNA extraction
and compared to the products after additional purification step involving DNase I. Products obtained after
different RNA extraction and purification methods were visualized using 2% agarose gel electrophoresis.
In conclusion, DNase I based RNA purification represents a necessary step for the assurance of a high-
quality extracted RNA used for gene expression analysis studies. Reliance on commercial kits for RNA
extraction only, without performing additional purification step can lead to errors in drawing final
conclusions and/or to false negative gene expression profiling, affecting the overall diagnostic procedure.
According to obtained results, the type of sampling used in this study was not suitable for the subsequent
gene expression analysis.