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S. Srinivasan, M. Senchyna, L. Heikkila, L. Jones; Method Optimization for the Isolation of Total RNA from Human Conjunctival Epithelial Cells Collected via Impression Cytology . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1500.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To optimize a technique for the isolation of total RNA derived from human conjunctival epithelial cells collected in vivo via impression cytology. Methods: Conjunctival epithelial cells were collected via impression cytology from human volunteers using either Millipore (MP) or Poly Ether Sulfone (PES) membranes. Following collection, total RNA was isolated using one of two commercially available methods: TRIzolTM (TZ) (Life Technologies) or RNeasyTM Mini (RN) (Qiagen). 100 µL of TZ reagent was used per membrane where as either 600 µL or 1.0 mL of lysis (RLT) buffer was used with the RN kit. RNA concentration and integrity (λ260:280) was assessed spectrophotometrically and via 1% agarose–formaldehyde electrophoresis. RT–PCR of mRNAs coding for MUC1 and the housekeeping gene GAPDH was performed to confirm the collection of high quality, DNA–free RNA. Results: As is described in Table 1, the RN method using of 600 uL of lysis buffer resulted in the inconsistent isolation of RNA that was also lower in quality and yield compared to the other techniques. When the TZ and 1.0 mL RN methods were compared, total RNA yield was greater with the MP membrane (p=0.05), however, both membranes consistently provided high quality (λ260:280 >1.8) RNA with no significant difference between the kits (p=NS). All RNA isolated with the TZ and 1.0 mL RN methods demonstrated positive amplification of MUC1 and GAPDH mRNAs as assessed by RT–PCR. Negative controls failed to produce amplification products. Conclusions:We conclude that MP membranes processed with either the TZ or 1.0 mL RN methods are equally efficient for the isolation of high quality RNA from conjunctival cells collected in vivo. From a "user friendly" view point, we recommend the adoption of the 1.0 mL RN method due to enhanced speed as well as on–column isolation and DNase digestion capabilities.
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