Nucleic acid storage on FTA cards from cytological samples

Dear Editor, In a recent article published in Cytopathology, we investigated the use of FTA cards to store DNA of lymphoid cells obtained by lymph node fine needle cytology (FNC). We proved the quality of DNA stored on FTA cards and its suitability for immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessments. We had obtained similar data using the DNA of breast cancer cells and these latter data were similar to other experiences. We continued the study to investigate whether RNA could also be stored and purified from the same samples on FTA cards. For this purpose, we performed RNA extraction from lymph node cells obtained by FNC from: 20 benign reactive (BRH), 20 nonHodgkin lymphoma (NHL) and 20 breast cancer (BC) cases. All the cases were partially stored on FTA cards at room temperature and partially cryopreserved. RNA extractions of FTA punches and cryopreserved cells of all the samples were carried out after a median of 18 months, using QIAamp RNeasy Micro Kit” (Qiagen, Valencia, CA, USA, cat. n. 74004), according to the manufacturer’s protocol (Figure 1). Cells were disrupted and homogenised by vigorous vortexing for FTA punches and by passing cryopreserved cells through a 20-gauge (0.9 mm) needle attached to a sterile plastic syringe for 5-10 times. RNA yield and quality were then quantified by the NanoDrop 1000 V 3.7. 1 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA); integrity was assessed by RT-PCR of a control gene composed of six genes producing fragments of 100200-300-400-600 bp, as previously described. RT-PCR was carried out using SuperScript VILO MasterMix (Life Technologies Corporation, Carlsbad, CA, USA, cat. n. 11755-500), according to the manufacturer’s protocol, to generate cDNA starting from a low amount of RNA input. Samples were run in duplicate to ensure PCR reproducibility in all the cases. The median values of RNA yield from cryopreserved samples were: 29.75 ng/lL (range: 7.8 ng/lL to 191.1 ng/lL) in BRH, 21.7 ng/lL (range: 9.9 ng/lL to 167.9 ng/lL) in NHL and 32.9 (range: 7.7 ng/lL to 78.8) in BC. The median OD ratios were: 1.81 (range: 1.50 to 1.98) in BRH, 1.80 (range: 1.50 to 2.10) in NHL and 1.70 (range: 1.50 to 1.90) in BC. Conversely, no RNA was obtained by FTA cards in 50 samples. In the remaining 10 cases (3 BRH, 5 NHL, 2 BC), the median values of RNA were 0.4 ng/lL (range: 0.15 ng/lL to 32.1) and the OD was 1.25 (range: 0.0 to 12.9), which are not valuable. The expected PCR bands of the control gene appeared in all the cryopreserved BRH, NHL and BC cases, but not in the FTA cards. Some previous studies have evaluated the possibility to store RNA from virus, mosquitoes, protozoa, trypanosome, plasmodia, bacteria and vegetable cells on FTA cards. ; these studies did not succeed in RNA storage on FTA cards. Just one study achieved this goal, but the quality of the RNA obtained was poor, especially when extracted after 8-12 months. As far as human RNA concerns, only two studies succeeded in extracting RNA from FTA cards stored cells. In the first study, 264 bone marrow cells or whole blood of Iraqi children with acute lymphoblastic leukaemia (ALL) were spotted on FTA cards and preserved at 4°C for up to 6 weeks before RNA extraction. In this case, the success of the procedure was probably as a result of the 4°C storage, which is not indicated by the manufacturer or probably related to the type of sample (ALL), which provided a large amount of RNA that compensated the losses. In the second study, the RNA of 66 ovarian carcinoma cells was extracted from FTA cards. In this study, RNA stored on FTA cards proved to be stable for 6 months only, while in our study, storage times ranged from 6 to 24 months. The weaknesses of FTA card RNA storage are probably due to the high fragmentation of RNA on this support; the early extraction or the 4°C storage only partially overcome the inadequacy of FTA cards for this specific goal. Notwithstanding this limitation on RNA storage, we believe that FTA cards remain a good alternative to store DNA for molecular analyses.