Stool Total RNA Purification Kit

Product Description

• Simultaneous isolation of both host RNA and microbial RNA (universal protocol)
• Eliminates PCR inhibitors including humic acid
• Fast and easy processing - Rapid isolation of high-quality, ready-to-use RNA in 30 minutes.
• No organic extraction or alcohol precipitation - Phenol is not required for the isolation
• High yields - Consistent, high yields of inhibitor-free RNA

Norgen’s Stool Total RNA Purification Kit provides a convenient and rapid method to purify total RNA from small amounts of stool samples. All types of stool samples can be processed with this kit, including animal fecal samples and manure. The kit removes all traces of humic acid using the provided Bead Tubes and a combination of chemical and physical homogenization and lysis. A simple and rapid spin column procedure is then used to further purify the RNA. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA and small interfering RNA. The protocol does not rely on the use of phenol or chloroform, thereby providing a user friendly procedure and allowing high-throughput analysis on the lab bench. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real time PCR and reverse transcription PCR for gene expression analysis.

The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real time PCR and reverse transcription PCR for gene expression analysis.

High Yields of Stool RNA Total RNA was purified from 200 mg of human stool using Norgen’s Stool RNA Purification Kit and a leading competitor’s kit, in triplicate. For analysis, 7.5 µL of each 75 µL elution was loaded on a 1.2 % 1x MOPS formaldehyde-agarose gel. Norgen’s kit was found to have a higher yield of RNA, isolated from 200mg of female human toddler stool.

Yield and Quality of Purified Stool RNA. Total RNA was isolated from 200 mg of human stool using Norgen’s Stool RNA Purification Kit and a leading competitor kit. Comparisons were then made based on yield, and A260:A280/A260:A230 ratios measured using the NanoVue Plus™. In Panel A it can be seen that both kits isolated RNA with high A260:A280 ratios (all samples were found to be above 1.8 and below 2.2). In Panel B, Norgen’s kit was found to isolate RNA with a high A260:A230 (with all samples once again falling in the 1.8-2.2 range). The competitor kit, however, was found to isolate RNA with extremely low A260:A230 ratios, with none of the samples displaying a A260:A230 ratio higher than 0.20. The resulst in Panel C are in agreement with the gel photo from Figure 1, and Norgen’s kit was found to isolate higher amounts of RNA, with an average yield of 14.58 µg, compared to the competitor’s average of 12.26 µg.

Detection of Bacterial Stool RNA using 16S Primers. Total stool RNA was isolated from 200 mg human stool samples using Norgen’s Stool RNA Purification Kit and a leading competitor’s kit. Five microliters of purified RNA was used in a 20 µL reverse-transcription reaction using Invitrogen’s Superscript III system with 16S reverse primers. The cDNA generated was then used in a qPCR reaction involving Norgen’s 2X PCR Mastermix spiked with SYBR green (Bio-Rad), using 0.3µM of primers against bacterial 16S. As can be seen in the amplification plot, Norgen’s kit outperformed the leading competitor’s kit by on average 1.5 Ct values. This indicates that Norgen isolated higher quality and yields of RNA from stool, that can be used in an array of downstream applications.