This application note describes the use of the Agilent RNA 6000 Pico assay for the assessment of genomic DNA contamination in total RNA isolated from embryonic kidney cells using laser capture microdissection (LCM). Integrity and relative concentration of total RNA were analyzed using the Agilent 2100 bioanalyzer and the RNA 6000 Pico assay. An unexpected peak between the 18S and 28S ribosomal RNA peaks was observed and unambiguously proven, by DNase digestion and genomic DNA spike-in experiments, to be caused by genomic DNA contamination. A simple on-column DNase digestion method is suggested to eliminate contaminating genomic DNA from total RNA samples obtained by LCM, fluorescent-activated cell sorting (FACS), or manual dissection.
This document details the use of the Agilent 2100 bioanalyzer with the RNA 6000 Pico assay to analyze total RNA isolated from mouse embryonic kidney cells via Laser Capture Microdissection (LCM). The study identifies an unexpected peak between 18S and 28S ribosomal RNA peaks as genomic DNA contamination.
The text validates this finding through DNase digestion and genomic DNA spike-in experiments. It concludes that an on-column DNase digestion method using the Qiagen RNeasy Mini kit effectively eliminates this contamination, improving RNA yield and integrity for downstream microarray experiments.
The unexpected peak is caused by genomic DNA contamination.
An on-column DNase digestion method during RNA purification can eliminate contaminating genomic DNA.
The Qiagen RNeasy Mini RNA extraction kit is recommended for cleanup.