Cell culture and formalin fixation
Nthy-ori 3-1 (ECACC, Wiltshire, UK) is a normal thyroid follicular epithelial cell line derived from adult thyroid tissue that has been transfected with a plasmid encoding for the SV40 large T gene .
This cell line was grown to confluence in a humidified atmosphere containing 5% CO2 at 37°C in the following plating medium: RPMI 1640 with 2 mM L-glutamine, 10% Foetal calf serum (FCS), Penicillin (100 U/ml) and Streptomycin (100 μg/ml). Tripsinized cells were counted with a hemocytometer. Suspended cells were aliquot and were pelleted (a) snap frozen and (b) formalin fixed and paraffin embedded into a cell block. When formalin fixation was required, a cohesive solid cell pellet was constructed using 20% agar. The cells were centrifuged in an eppendorf tube, and the supernatant was removed using a pipette. Approximately 30 μl of pre-warmed agar (60°C) was added to each tube. The solid cell pellet was formed within a few seconds. Cell blocks were fixed following standard tissue processing which included 10% buffered formalin fixation for 4 hours, on a Tissue-Tek® V.I.P.™ tissue processor. The pellets were subsequently paraffin embedded.
RNA was extracted from fresh cells using mirVana™ miRNA Isolation kit (Ambion Ltd., Cambridgeshire, UK) and from FFPE cells using RecoverAll™ Total Nucleic Acid Isolation kit (Ambion Ltd., Cambridgeshire, UK) following the manufacturer's protocol. For snap-frozen extraction, one extraction was performed using approximately 4 × 105 cells. For FFPE extraction, 4 extractions were performed in parallel with one pellet (1 × 106 cells) in each extraction. The entire pellet was dissected from each block and was finely minced using a scalpel. These preparations were then deparaffinized, followed by proteinase K digestion for 3 hours at 50°C, on column DNase digestion and elution as described in the protocol. At that point all 4 FFPE extracts were combined into one tube designated the FFPE sample. The concentrations of these two samples were measured using a NanoDrop® ND-1000 Spectrophotometer (Wilmington, USA) and extracts were diluted to 10 ng/μl. RNA quality was measured using the RNA 6000 Pico LabChip® Kit on an Agilent 2100 Bioanalyser (Agilent technologies, Waldbronn, Germany).
Applied Biosystems TaqMan® microRNA (miRNA) assays (designed for mature miRNA quantification using Applied Biosystems Real Time PCR instruments) were utilised in this study. The human panel early access kit (P/N: 4365381, Applied Biosystems) used in this study contained 160 individual assays and comprised two steps: Reverse Transcription (RT) and real time PCR. The stem-loop RT primer specifically hybridizes to a miRNA molecule and is reverse transcribed with a MultiScribe reverse transcriptase . The RT products are then quantified using real-time TaqMan® PCR.
Applied Biosystems High-Capacity cDNA Archive Kit (P/N: 4322171, Applied Biosystems, CA, USA) was used following manufacturer's protocol for reverse transcription. Each RT reaction contained 50 ng of extracted total RNA, 50 nM stem-looped RT primer, 1 × RT buffer, 0.25 mM each of dNTPs, 3.33 U/μl Multiscribe reverse transcriptase and 0.25 U/μl RNase Inhibitor. The 15 μl reactions were incubated in an Applied Biosystems Thermocycler in a 96-well plate for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and then held at 4°C.
For the Real-time PCR step, amplification was carried out using sequence specific primers on the Applied Biosystems 7900 HT Real-Time PCR system. The 20 μl reaction included 1.33 μl RT product, 1 × TaqMan® Universal PCR Master Mix with no UNG (P/N: 4324018, Applied Biosystems) and 1 × TaqMan® MicroRNA assays. The reactions were incubated in a 96-well optical plate at 95°C for 10 min, following by 40 cycles of 95°C for 15 s and 60°C for 1 min. The real-time PCRs for each miRNA were run in triplicate. hsa-let-7a was included as an endogenous control and cel-lin-4 was incorporated as a negative control.
Replicates were omitted if Ct standard deviation was greater than 1.5. All 160 miRNAs were detectable with the exception of c-lin-4 in FFPE and c-lin-4, mir-104, mir-122a, mir-144, mir-302b and mir-325 in snap frozen sample. The data was collected using Microsoft Excel and was statistical analyzed using MINITAB® 14 on ΔCts with the formulas below:ΔCt = Ct_Mean(FFPE) - Ct_Mean(Snap frozen)ΔΔCt = ΔCt - ΔCt_MeanExpression level = 2-ΔΔCt