Optimization of DNA Extraction from Low-Yield and Degraded Samples Using the BioRobot^�EZ1 and BioRobot^�M48

Abstract

Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots EZ1 or BioRobots M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1.

Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer

“…The addition of poly(A) carrier RNA but not glycogen previously increased the recovery of automated silica-based extractions (BioRobots EZ1 and BioRobots M48, Qiagen) by an average of 24% (23). In another paper, a five-fold increased recovery was obtained in DNA extractions carried out on silica-based monoliths within a microfluidic device when poly(A) carrier RNA was added to the chaotropic salt solution (22).…”

Comparison of an automated nucleic acid extraction system with the column-based procedure

“…First successful applications of the EZ1 system, which requires considerable less handson time of technical laboratory assistants than tradition column-based extraction systems [23], were published in the fi elds of microbiology [24] and forensic medicine [25,26] as early as in 2005. The EZ1 system was shown to be useful for the isolation of target DNA in low quantities, e.g., of fetal DNA in maternal plasma [21], or of DNA in partially degraded biological specimens [22]. Successful EZ1-based DNA extraction from formalin-fi xed, paraffi nembedded tissue samples [9], or otherwise long-term preserved tissue specimens [8] has been demonstrated.…”

Evaluation of collection and extraction methodologies of latent fingerprints for military application

“…DNA was isolated by two methods: a) Lysis Solution Extraction (LSE) [4]: samples were extracted using a 30 min incubation with 0.05% SDS and proteinase K at 56 C, followed by incubation at 100 C for 10 min. Concentration of the extract and removal of the SDS was accomplished through centrifugation (Amicon1 100 column, Millipore) pretreated with 1ng of Poly A RNA and b) QIAmp1 DNA Investigator1 Kit (Qiagen), according to the manufacturer's instruction.…”