Human blood from one donor was treated with sodium citrate, heparin, or EDTA. Genomic DNA was purified from a 50 or 200 µl aliquot of the treated blood manually with the QIAamp 96 DNA Blood Kit or on the QIAcube HT using the QIAamp 96 DNA QIAcube HT Kit with the QIAamp 96 DNA QIAcube HT V1.QSP protocol. Purified DNA was eluted in 200 µl. PCR amplification of a 1.2 kb fragment from the single copy Hugl gene was carried out using QIAGEN HotStarTaq DNA Polymerase and 2.5 µl eluate in a total volume of 25 µl. A 1 µl aliquot of each PCR was analyzed on the QIAxcel Advanced with the QIAxcel DNA Screening Kit. M1: pUC18 HaeIII marker; M2: phiX174 HaeIII marker.
RNA was isolated from 10 mg rat tissue (lung, kidney, heart, brain, spleen, and intestine) stabilized in RNAlater RNA Stabilization Reagent. Sample processing was automated on the QIAcube HT, BioRobot Universal System, or the QIAcube using QIAzol Lysis Reagent. A 1 µl aliquot of each purified RNA sample was analyzed on the QIAxcel Advanced using the QIAxcel RNA QC Kit v2.0 and the QX RNA Size Marker 200–6000 nt. The resulting high RNA integrity scores (RIS) indicate recovery of high-quality RNA.
Viral nucleic acids were purified from 200 µl plasma samples spiked with 10,000, 1000 and 100 IU/ml of a typical DNA or RNA virus. Sample processing was automated using either QIAcube HT with the QIAamp 96 Virus QIAcube HT Kit and protocol, QIAxtractor with the VX Protocol, or QIAcube with the QIAamp MinElute Virus Spin Kit. Viral nucleic acids were detected using in-house PCR and RT-PCR assays, with 20 µl eluate per reaction on the Rotor-Gene Q. Results show that QIAcube HT with the QIAamp 96 Virus QIAcube HT Kit performs as well as or better than the other methods.
Various sample types were spiked with 20,000 IU of a typical DNA virus. Viral DNA was purified from 200 µl of each sample lysate using the QIAcube HT with the QIAamp 96 Virus QIAcube HT Kit and protocol. Purified viral DNA was detected using an in-house PCR assay with 20 µl each eluate per reaction.
Aliquots of 200 mg of three different human stool specimens were processed on the QIAcube HT using the QIAamp 96 PowerFecal QIAcube HT Kit, on the QIAcube using the QIAamp DNA Stool Mini Kit and QIAamp Fast DNA Stool Mini Kit, or manually using the two DNA Stool kits. Real-time PCR was carried out on the Rotor-Gene Q using the QuantiTect Virus PCR Kit with 2 µl and 10 µl eluates respectively, from each sample in 20 µl reactions containing an internal control. CT values were compared to a standard containing no sample but only internal control (dark blue bar). All the three samples either extracted on different instruments or manually showed similar performance compared to the standard, regardless of the volume of eluate used, indicating no PCR inhibition.
Aliquots of 200 mg stool sample from different animals were processed on the QIAcube HT instrument (blue bars) using the QIAamp 96 PowerFecal QIAcube HT Kit, or manually using the QIAamp Fast DNA Stool Mini Kit (pink bars). Real-time PCR of 16S rRNA gene of Corynebacterium glutamicum was carried out on the Rotor-Gene Q using the QuantiFast Probe PCR Kit with 1 µl eluate in a 20 µl reaction. DNA extracted either manually or on the QIAcube HT instrument showed similar performance for all the stool samples.
Consistent amounts of BVDV particles and Salmonella cells were simultaneously spiked into a variety of animal specimens. Samples were processed in parallel on the QIAcube HT with the cador Pathogen 96 QIAcube HT Kit or manually with the QIAamp cador Pathogen Mini Kit. Co-isolated viral RNA and bacterial DNA were identified using an in-house assay. Mean CT values are shown for [A] BVDV and [B]Salmonella. Small error bars (±1 SD of 3 extraction replicates) indicate high precision for both purification procedures.
The DNeasy mericon 96 QIAcube HT Kit has been adapted for rapid mid- to high-throughput purification of DNA from a variety of raw and processed food matrices on the QIAcube HT instrument. The modified cetyltrimethylammonium bromide (CTAB) extraction procedure allows DNA extraction from food samples in radically shorter time compared with conventional CTAB protocols.
Blood samples of various species were spiked with BVDV particles and were frozen at –20°C. Of each sample, 4 replicates were processed on QIAcube HT with the cador Pathogen 96 QIAcube HT Kit in 3 subsequent runs on different days. [A] Viral RNA was identified using an in-house assay, which included [B] an internal amplification control. Error bars represent ±1 SD of 3 replicates. The BVDV CT values for each set of quadruplicates showed a mean relative standard deviation of 0.67 ± 0.43%, and the inter-assay CV for each sample from 3 runs ranged from 0.14 to 0.89% (mean: 0.45%), indicating high repeatability of viral RNA purification on QIAcube HT.
Genomic DNA was purified from 200 µl human blood. Samples were processed with the QIAcube HT using the QIAamp 96 DNA QIAcube HT Kit and the QIAamp 96 DNA QIAcube HT V1.QSP protocol, or with the QIAcube using the QIAamp DNA Blood Mini Kit. Quantitative PCR of IL8 was carried out using the QuantiFast Probe Kit with 5 µL eluate in 25 µl. The CT values obtained show even amplification across the whole 96-well plate, indicating no well-to-well variation in DNA recovery with QIAcube HT. CT values were similar between the different purification platforms.
Nucleic acids were purified from serial dilutions of plasma samples spiked with a typical RNA or DNA virus. Samples were processed using QIAcube HT with the QIAamp 96 Virus QIAcube HT Kit and protocol and were analyzed using in-house PCR and RT-PCR assays. The percentage of positive samples at low virus titers is shown. The resulting 95% probit values were 316.84 IU/ml for the RNA virus and 18.54 IU/ml for the DNA virus.
Lysates from rat lung tissue stabilized in RNAlater were serially diluted to produce samples containing equivalents of 2.5–25 mg tissue. Total RNA was purified from these samples using the QIAcube HT with the RNeasy QIAcube HT Kit and the RNeasy 96 QIAcube HT total RNA Tissue V1.QSP protocol. Purified RNA was eluted in 100 µl RNase-free water. Optical density measurements show a linear relationship between RNA recovery and amount of starting material.
DNA was purified from orange juice, chocolate, and peanut butter using the QIAcube HT instrument and the DNeasy mericon 96 QIAcube HT Kit or manually using the DNeasy mericon Food Kit. Purified DNA was analyzed by qPCR of the trnL chloroplast tRNA gene using the QuantiFast SYBR® Green PCR Kit. The resulting CT values indicate similar performance from both purification methods. Diluting the input DNA 1:10 for qPCR analysis shows that all purified DNA was also free of PCR inhibitors.
Genomic DNA was purified from 5 mg rat lung, kidney, spleen, and liver tissue and 200 µl human blood. Samples were processed with the QIAcube HT using the QIAamp 96 DNA QIAcube HT Kit and the QIAamp 96 DNA QIAcube HT V1 QSP protocol or with the QIAcube using the QIAamp DNA Blood Mini Kit (blood) or the QIAamp DNA Mini Kit (tissues). Both platforms showed similar performance in [A] yield and [B] quality of purified DNA for each type of starting material.
RNA was purified from 5 x 105 Jurkat cells on the QIAcube HT using the RNeasy 96 QIAcube HT Kit with the RNeasy 96 QIAcube HT total RNA cell with DNase V1.QSP protocol. Purified RNA was eluted in 110 µl RNase-free water. Quantitative RT-PCR of the human β-actin mRNA was carried out using the QuantiFast Probe RT-PCR Kit with 5 µl eluate in a total volume of 25 µl. The CT values obtained throughout one 96-well plate show even amplification across the whole plate, indicating no significant well-to-well variation in RNA recovery.
