Allprep dna rna isolation kit

Two replicate swabs from each of the six sites were thawed on ice and placed together into a sterile Lysing Matrix E tube (MP Biomedicals, Australia). Genomic DNA was extracted from the samples using the AllPrep DNA/RNA Isolation Kit (Qiagen) following the manufacturer’s instructions and eluted in 30 μL of DNase-free water. Cells were ruptured using a Qiagen TissueLyser II at 25 m/s for 2 × 40 s. The quality and quantity of genomic DNA were measured on a Nanodrop 3300 fluorospectrometer and by using PicoGreen (Quant-iT dsDNA kit, Invitrogen) dye.

qPCR was used to measure the abundance of B.thetaiotamicron in CD19^−/− mice colonized with isogenic B.theta strains (see description below). 40 days post-colonization, fecal pellets were collected, snap frozen in liquid nitrogen, and stored at −80 °C until use. Fecal DNA was extracted using the Allprep DNA/RNA Isolation Kit (with a 3 min bead beating step) (Qiagen). Sample DNA concentration was measured using a Nanodrop Spectrophotometer. Total bacteria were estimated using universal 16S qPCR primers (338F/518R), and B.theta abundance was estimated using B.theta-specific primers (Supplementary Fig. S13). Ct values were converted into amplicon copies using the following equation 10^{((Ct-35)/−3)}. Amplicon copies were standardized to the amount (nanograms) of DNA added to each reaction.  qPCR was performed on a CFX Real-Time qPCR Instrument (Bio-Rad) using the iTaq Universal SYBR Green Supermix (Bio-Rad). Primer sets used in qPCR experiments are provided in Supplementary Table 2.

Two secondary endpoint urine detection assays (Veterinary-Bladder Tumor Antigen Test, V-BTA; and a BRAF^V595E mutation detection assay) were performed on midstream, free catch urine samples. The V-BTA test (PolyMedco Inc. Seattle, Washington, test kits provided by PolyMedco), a rapid latex agglutination urine dipstick test, was performed according to the manufacturer protocol (52 (link)) on urine samples as they were collected, and the results were analyzed at the end of the screening study. The presence of the BRAF^V595E mutation was analyzed in urine sediment samples which were snap frozen and stored at -80°C over the course of the study, and the samples were run in batch at the end of the screening study. Briefly, DNA was isolated using the AllPrep DNA/RNA isolation kit (Qiagen, Germantown, MD), the presence of the BRAF^V595E mutation determined by a droplet digital PCR (ddPCR) assay, and the mutation fraction, i.e., the percentage of alleles with the mutation, was determined as previously described (36 (link), 37 (link)). Linearity in the assay was established for wild type and mutant DNA, and was preserved below 0.5 copies/µl. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the presence of existing disease or subsequent bladder cancer development were calculated.

One lozenge from each of groups A and B was dissolved separately in 1 mL of sterile, PCR-grade water. Aliquots of the clinical samples and the dissolved lozenges were mechanically lysed using Lysing Matrix E bead tubes and RLT Plus lysis buffer for 30 s × 2 using the Omni Bead Ruptor 24. Purification of genomic DNA was achieved with the AllPrep DNA/RNA Isolation Kit (Qiagen, NRW, Germany), using a spin column for DNA isolation. PCR-grade water was used as a negative extraction control.
The V3-V4 region of the bacterial 16S rRNA gene was amplified using Illumina-compatible primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21^{35 (link),} as previously described^{32 (link)}. Duplicate PCR reactions were pooled and purified for sequencing, as previously described^{29 (link)}. Normalised samples were submitted to Auckland Genomics Ltd for sequencing on the Illumina MiSeq platform.

mRNA was isolated using RNeasy mini kit (Qiagen) and reverse transcribed by reverse transcription system (Promega) according to manufacturer’s instructions. Real time PCR was performed using an Applied Biosystem 7500 System and primers and probes for claudin-5, ZO-1 and occludin (ThermoFisher). GAPDH mRNA level was assessed in duplication for data normalization.
Tissue detection of EcoHIV/NDK was performed 1 week post infection. Tissue was harvested and processed using All Prep DNA/RNA isolation kit (Qiagen). RNA was reverse transcribed as described above. HIV detection was then conducted using the following primers and probe: NDKgag_F 5′-GACATAAGACAGGGACCAAAGG; NDKgag_R 5′-CTGGGTTTGCATTTTGGACC; NDKgag_Probe 5′-AACTCTAAGAGCCGAGCAAGCTTCAC. Normalization was conducted based on GAPDH for RNA or mouse HBB for DNA80 (link).