Nucleic Acid Isolation for Microbiome Research

Automated or manual recovery of high-quality nucleic acids from challenging samples such as stool and soil ready for downstream processing in microarrays, real-time PCR, and next-generation sequencing. Lysis of microbial samples using a 96-well bead plate.

Explore: MagMAX Microbiome Ultra Nucleic Acid Isolation Kit

Automated or manual recovery of high-quality nucleic acids from challenging samples such as stool and soil ready for downstream processing in microarrays, real-time PCR, and next-generation sequencing. Lysis of microbes using lysis bead tubes.

Explore: MagMAX Microbiome Ultra Nucleic Acid Isolation Kit

Figure 1. Quality comparison of purified nucleic acids using the MagMAX microbiome kit. Quality of total microbial nucleic acid purified from feces (100 mg input) with MagMAX Microbiome Ultra Nucleic Acid Isolation kit was analyzed on a 1% agarose gel. 1 μg of total nucleic acid sample from each donor was loaded per lane in duplicate for six human donors. Clear bands of both genomic DNA and RNA can be seen on the ethidium bromide pre-stained agarose gel. The Invitrogen 1 Kb Plus DNA Ladder was utilized for size determination. (Note: Since this is not a denatured gel, RNA sizes shown are different from actual. Gel analysis was performed under native conditions, and thus RNA sizes appear somewhat different from the actual size.)

Figure 2. Yield comparison of purified nucleic acids using the MagMAX microbiome kit. Total Nucleic acid yields (both DNA + RNA) isolated from fecal (100 mg input) samples collected from six human donors were measured on Nanodrop 2000.

Figure 3. Bioanalyzer trace of human fecal total RNA isolated using a bead tube version of MagMAX Microbiome Ultra Nucleic Acid Isolation kit. RNA 6000 Nano Kit with the Prokaryotic RNA assay was used to analyze the quality. Representative electropherogram indicate the position of 16S and 23S rRNA peaks. Unlabeled peaks correspond to additional ribosomal RNA.

Figure 6. qPCR analysis of DNA purified from fungal cultures with the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. qPCR was performed using a total fungal TaqMan assay. TaqMan Fast Advanced Master Mix was used under fast cycling conditions.

Figure 7. qPCR analysis of DNA purified from human donors with C. difficile infection using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. Samples were obtained from Discovery biosciences and qPCR was performed using a C. difficile TaqMan assay. TaqMan Fast Advanced Master Mix was used under fast cycling conditions.

Figure 8. qPCR analysis of total nucleic acid isolated from Texas soil samples with the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. TaqMan Assays were performed for a mixed gram positive and gram-negative target (16S) and a Gram-negative target (Bacteroidetes). Twenty-fold dilution of soil total nucleic acid preps was used for TaqMan assays. For RNA analysis, total nucleic acid was treated with DNase and reverse transcribed using Superscript Vilo Master Mix to make the cDNA. TaqMan Fast Advanced Master Mix was used under fast cycling conditions.

Figure 9. Species level 16S profile for microbial DNA purified from stool of a human donor with ulcerative colitis (UC). Total nucleic acid was purified from the stool of a patient with UC (in duplicates) and 16S Metagenomics kit and Ion plus fragment library kit were used to synthesize 16S libraries. The barcoded libraries were pooled and templated on Ion Chef Instrument followed by sequencing on the Ion S5 System. Automated analysis, annotation, and taxonomic assignments were performed on Ion reporter software. A fecal sample DNA from a healthy donor is shown for species level comparison and abundance scale is shown above the profile. Red being the highly abundant species and blue being not detected/rare. R studio program is used to generate heat maps using log (2) values.