Rapid Automated DNA/RNA Extraction: Validation of Quick Gene 810 (QG) for Blood and Tissue Specimen Processing
M Cankovic, J Beher, L Whiteley, RJ Zarbo, D Chitale. Henry Ford Hospital, Detroit, MI
Background: Molecular Pathology is a rapidly growing area offering many tests. Testing is performed on a variety of samples such as peripheral blood, bone marrow, formalin fixed paraffin embedded (FFPE) tissue etc. Therefore there is a growing need for automation, high throughput, random access in method of nucleic acid extraction. We report our experience with the Fuji Film QG, a bench-top DNA/RNA isolation instrument (AutoGen,Holliston,MA).
Design: QG and manual DNA extraction protocols (QIAampDNA Mini Kit, Qiagen, Valencia,CA) were run in parallel for genomic DNA from 12 blood specimens (WBC 4.9-9.9 x 106/mL) and 12 FFPE tissues-colon(3), breast(2), lung (2), prostate(2), kidney(1), skin(2). QG and manual protocols (5 Prime,Gaithersburg,MD) were used to isolate total RNA from blood and the QG and TrimGen Wax Free (TrimGen,Sparks,MD) from FFPE tissues. DNA/RNA quantity were evaluated by spectrophotometry. DNA integrity was assessed by amplification of Control Size Ladder mix (InVivo Scribe,San Diego,CA) generating a series of amplicons. RNA integrity was assessed by real-time quantitative reverse transcription PCR by measurement of expression of 2 microglobulin (B2M) transcripts, with acceptable cycle threshold (Ct) values of 30 for blood and 35 for FFPE tissues run in duplicates.
Results: Extraction times were shorter by QG for Blood DNA/RNA. Manually isolated FFPE DNA was of superior quality. Both methods gave amplifiable DNA from blood and FFPE. Blood RNA from both methods was amplifiable, but the manually extracted samples gave lower Ct values. For tissue RNA, Ct was at background for 10/12 samples for QG, compared to Ct 24.95 to 32.39 for manual extraction.
|DNA extraction time||10 min||40 min||60 min||74 min|
|Mean DNA purity||1.75(1.63-2.0)||1.71(1.42-1.93)||1.10(0.69-2.81)||2.05(1.5-2.3)|
|RNA extraction time||39 min||45 min||60 min||74 min|
|Mean RNA purity||1.46(0.74-2.72)||1.19(0.75-2.46)||0.26(0.00-3.85)||1.15(1.0-1.3)|
|RNA integrity||Fair||Good||Failure 10/12||Good|
Conclusions: QG was easy to incorporate into the lab and is suited for small to medium throughput laboratories. The greatest time and cost savings were with DNA isolations from blood (30 minutes and $15 /sample). Blood RNA and tissue DNA protocols didn't yield substantial time savings, but were still cost effective due to elimination of several manual steps. Tissue RNA quality was suboptimal with QG in our experience.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 238, Wednesday Afternoon