Troubleshooting of RNA isolation methods in Papanicolaou HPVinfected smears

Cervical cancer is one of the leading causes of cancer in women, worldwide. Infection with humanpapillomavirus (HPV) has been accepted as the primary cause for the development of invasive cervicalcancer and its precursor lesions. Despite HPV infection has been proposed as an indispensable factor forcervical cancer development, only a subset of neoplastic lesions with HPV infection persist and progress toinvasive cancer. This suggests us that other molecular events are also involved in cancer progression. Aimof this study was to extract mRNA from cytobrush-collected healthy and HPV infected cervical epithelialcells and investigate various RNA extraction and purification protocols for assessment of RNA yield andquality. Taking into consideration that cervical cancer screening is based on the cytology basedPapanicolaou test (Pap test), main challenge is to investigate whether the samples obtained by regular Paptesting can be used for gene expression analysis. For this purpose, a total of 68 cervical specimens werepreviously tested for HPV infection. Following HPV testing, samples were submitted to RNA extractionand compared to the products after additional purification step involving DNase I. Products obtained afterdifferent 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 RNAextraction only, without performing additional purification step can lead to errors in drawing finalconclusions 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 subsequentgene expression analysis.