Influence of deep-freezing and MGG staining on DNA and RNA quality in different types of lung adenocarcinoma cytological smears.

Abstract

Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs). DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis. Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis. Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p< .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p< .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p< .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.