We are trying to extract HPV DNA from cervical swabs (ThinPrep) using MagNA Pure 96 equipment from Roche and we are encountering a serie of dificulties.
We have tried several approaches regarding sample preparation:
- 2 ml or 1 ml of cell suspension, wash with PBS, external lysis (1 hour or overnight incubation with Prot. K), take 500 ul directly to the equipament -> 50 or 100 ul of elution
- 500 ul of cell suspension directly to the equipment, without any sample preparation
- 500 ul of cell suspension directly to the equipament, without any sample preparation, and adding 50 ul of PBS 10x
We have used several protocols: Viral Universal, Pathogen Universal, DNA Cells, Viral NA Plasma.
The best results obtained were an agreement rate of 50% with the current extraction: manual protocol using Virus MinElute Kit from QIAGEN. The amplification system is a real time PCR with SYBR Green (Master Mix from Applied Biosystem) or a commercial kit INNO-LIPA from Fujirebio.
Does anyone have a tip to improve this agreement rate to 90 or 100%?
For other types of samples the results are good (plasma, urine, dried swab, LCR) except for the fact that in External Quality Assessment programs the extraction efficiency is very low: the diference in the Ct values are as big as 6 units.