Materials and methods
Cell culture and fixation
Human thyroid papillary carcinoma-derived K1 cells were obtained from DS Pharma Biomedical Co. Ltd. (Osaka, Japan), and maintained according to the manufacturer’s instructions. Briefly, the cells were cultured in a mixture of Dulbecco’s Modified Eagle’s Medium (Wako, Osaka, Japan), Ham’s F12 nutrient mixture (Wako), and MCDB105 medium (Sigma-Aldrich, St. Louis, USA) (2:1:1) supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 and 95% air. These cells were suspended in 5 ml CytoRich-Red and/or CytoRich-Blue fixative solutions (Becton–Dickinson, Franklin Lakes, USA), commercial products developed for LBC, and stored at 4 °C for 3 days.
Thyroid FNAB specimens
Five papillary thyroid carcinoma cases were randomly selected for this study. We obtained written informed consent and approval for this study from the Ethics Committee of Kuma Hospital (local IRB number: 20130808-1). The aspirated thyroid cells in the needles were transferred to CytoRich-Red or CytoRich-Blue fixative solutions.
Cell recovery and RNA isolation
K1 cells and the thyroid FNAB specimens suspended in CytoRich fixative solutions were obtained by trapping using Whatman GF/C glass-fiber filters (GE-healthcare, Little Chalfont, UK) (Additional file 1: Figure S1A). The filters with the trapped cells were washed with PBS and incubated in 500 μl lysis buffer [10 mM Tris–HCl (pH 7.8), 5 mM EDTA and 0.5% SDS] with or without 400 mg/ml Proteinase K (Wako) at 55 °C. After the removal of filters, the cell lysis solution was mixed with 1.5-ml RNAiso Blood (Takara-bio, Kusatsu, Japan), and subjected to RNA isolation according to the manufacturer’s instructions. Isolated RNAs were dissolved in 11 μl RNase/DNase-free distilled water, and RNA concentrations were determined using Quanti Fluor RNA System (Promega, Madison, USA) and the Fluorescence Spectrophotometer F-7000 (Hitachi, Tokyo, Japan).
RNA quality assessment
The isolated RNA was analyzed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, USA) in combination with the RNA 6000 Nano LabChip kit (Agilent), and its quality was assessed by RNA integrity number (RIN).
Real-time reverse-transcription PCR
For the quantitative reverse-transcription (RT)-PCR analysis of U6 small nuclear RNA (RNU6) levels, TaqMan MicroRNA Assays (Thermo-Fischer, Waltham, USA) were used. Monitoring of RNU6-derived PCR products was performed on a DICE Real-Time PCR System (Takara-bio).
Results and discussion
Application of LBC specimens to molecular diagnosis has been attempted in previous studies. Most of these studies, however, focus on genomic mutations, and studies on RNA analysis are limited. To our knowledge, the only study to evaluate RNA expression using FNAB specimens was conducted in medullary thyroid carcinomas [16]. In that study, the mRNA expression in leftover cells in needles was analyzed by conventional RT-PCR. Therein, the cells were directly put into cell lysis/denaturation solution, possibly leading to minimized RNA degradation and subsequent successful mRNA detection. However, it is difficult to perform this procedure routinely at a clinical site, mainly because of shortage of time and personnel. It would be convenient if residual LBC specimens could be used for RNA analysis after histopathological examination; however, this has not been achieved so far, possibly owing to the following reasons. Firstly, the cells in the alcohol-containing fixatives (CytoRich-Red; 23.3% isopropyl alcohol and 10% methanol, CytoRich-Blue; 44.0% ethanol and 5.0% methanol) are not efficiently recovered by simple centrifugation, since pelleted cells are rather crumbly, which complicates the removal of the supernatant. Another problem could be that RNA molecules are not efficiently recovered from the fixed cells, particularly those fixed with formaldehyde-containing fixatives that form intramolecular crosslinks, which prevent the release of RNA from insoluble cellular components.
To overcome the first problem, we tried to use a glass-fiber filter for cell tapping. The device for trapping cells in a glass-fiber filter is illustrated in Additional file 1: Figure S1A. After the K1 cell suspension was passed through the glass-fiber filter, only few cells were observed in the filtrate, indicating that the cells were efficiently trapped in the filter (Additional file 1: Figure S1B). Next, we evaluated the effect of Proteinase K treatment on RNA yields from the filter-trapped fixed K1 cells, because this treatment is known to improve RNA recovery from formalin-fixed paraffin-embedded specimens [14]. As shown in Fig. 1a, RNA yields were pronouncedly increased by treating the filter with CytoRich-Red-fixed K1 cells in a cell lysis buffer containing Proteinase K. Interestingly, this treatment was also effective on RNA recovery from K1 cells fixed with CytoRich-Blue (Fig. 1b), which does not contain formaldehyde. Thus, it is conceivable that Proteinase K treatment facilitates RNA recovery from rigid cells after dehydration. The results also show that RNA molecules were largely released from the fixed cells even after 1-h treatment. The RNA samples isolated from CytoRich-fixed cells were then subjected to quality assessment using Agilent 2100 Bioanalyzer. The RNA samples recovered from Proteinase K-treated fixed cells were shown to have an RIN of more than 9 (Fig. 1c, d), indicating that RNA integrity was well maintained during the procedure.
CytoRich-Red is more generally used than CytoRich-Blue for LBC of thyroid FNAB specimens, because CytoRich-Red efficiently lyses contaminated blood cells. Thus, only CytoRich-Red was used for the following experiments. To verify the importance of Proteinase K in RNA extraction, we extracted RNA from CytoRich-Red-fixed K1 cells incubated in both Proteinase K-containing and Proteinase K-free lysis buffers. As shown in Fig. 2, Proteinase K treatment pronouncedly improved RNA recovery from the sample, compared to poor recovery in the absence of Proteinase K. Then, we examined whether this finding is applicable to RNA extraction from clinical LBC samples. Five thyroid FNAB specimens fixed with CytoRich-Red solution were processed for RNA extraction in the presence or absence of Proteinase K. As expected, larger amounts of RNA were extracted from all these specimens in the presence of Proteinase K than in the absence of Proteinase K (Fig. 3).
To examine whether the isolated RNA was suitable for RNA-based molecular diagnostics, the RNA samples isolated from the CytoRich-Red-fixed K1 cells and thyroid FNAB specimens were subjected to quantitative RT-PCR to detect RNU6 RNA, a ubiquitously expressed small nuclear RNA. As expected, RNU6 RNA was detected in RNA extracted from the Proteinase K-treated materials (Additional file 1: Figure S2). The RNA samples extracted from the Proteinase K-untreated materials were not analyzed for RNU6 expression as RNA yields were too low (Figs. 2, 3). Reaction without a cDNA template (negative control) gave no signal (Additional file 1: Figure S2).
Overall, our data suggest that RNA can be isolated efficiently from thyroid cells fixed with LBC solutions. LBC is also used in the diagnosis of other cancer types such as gynecological cancers [17]; thus, the method developed herein could be more widely applicable.
