Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples.
BACKGROUND
This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies.


METHODS
A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific TP53 amplicons and TP53 quantitative reverse-transcribed real-time PCR (qRT-PCR)], and mRNA quality (functional analysis of separated alleles in yeast).


RESULTS
Although spectrophotometry and fluorometry results were comparable for both homogenization methods, TP53 expression values obtained by amplicon densitometry and qRT-PCR were significantly and consistently better after automated homogenization (p<0.005) for both uvula and tumor samples. Functional analysis of separated alleles in yeast results was better with the automated technique for tumor samples.


CONCLUSIONS
Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization.
Van der Vorst
Sébastien
S
Center for Applied Molecular Technology, Université catholique de Louvain, Brussels, Belgium.
Dekairelle
Anne-France
AF
Irenge
Léonid
L
Hamoir
Marc
M
Robert
Annie
A
Gala
Jean-Luc
JL
D006801:Humans; D009061:Mouth Mucosa / Q000473:pathology*; D009062:Mouth Neoplasms / Q000175:diagnosis*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods; D013064:Speech Disorders / Q000601:surgery; D046509:Tissue Culture Techniques / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D014609:Uvula / Q000601:surgery
D016159:Tumor Suppressor Protein p53; D012313:RNA