Qiagility liquid handling robot

Manufactured by Qiagen

The QIAgility liquid handling robot is a versatile laboratory instrument designed for automated liquid handling tasks. It features precise pipetting and dilution capabilities to streamline various sample preparation and assay setup processes. The QIAgility is a compact and user-friendly robotic system intended to enhance workflow efficiency and reproducibility in research and diagnostic laboratories.

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7 protocols using qiagility liquid handling robot

Inflammatory Gene Expression in Mouse Brains

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Fresh brains were collected for qPCR to evaluate inflammatory gene expression. The cortex was processed for RNA isolation using the RNeasy mini kit (Qiagen, Hilden, Germany) and a QIAcube (Qiagen), as per the manufacturer’s protocol. Eluted RNA concentration and purity were determined using a Qiagen QIAexpert spectrophotometer. One microgram of eluted RNA from each sample was reversed transcribed into cDNA using a QuantiTect Reverse Transcription kit (Qiagen) and diluted to 1:10.
Samples were pipetted into a 192-well plate using a Qiagility liquid handling robot (Qiagen). Samples were run in duplicate, and cDNA was amplified with a QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). TaqMan fast advanced gene expression assays (Thermo Fisher Scientific) were used for quantification of gene expression, as outlined in Supplementary Table S3.
Relative gene expression ratios were calculated using the 2^−ΔΔCT method and normalized to the geometric mean of the housekeeping genes (Ppia and Hprt), which we have previously shown to be stable in the brains of C57BL/6 mice [47 (link)]. The expression of each gene was normalized to control mice.

Chu E., Mychasiuk R., Tsantikos E., Raftery A.L., L’Estrange-Stranieri E., Dill L.K., Semple B.D, & Hibbs M.L. (2023). Regulation of Microglial Signaling by Lyn and SHIP-1 in the Steady-State Adult Mouse Brain. Cells, 12(19), 2378.

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Amplicon Library Preparation and Sequencing

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Amplicon library preparation, cleaning, and dilution were performed by the Microbial Analysis, Resources, and Services
(MARS) Facility at UConn. The V4 region of the 16s rRNA gene was amplified in triplicate using the dual barcoded primers (515F and
806R) of Caporaso et al (8 basepair Golay-barcoded indices on the 3’ end)(17 (link)) and Kozich et al (8 basepair on the 5’ end)(18 (link)).
Amplification was performed using 30 ng of DNA and AccuPrime Taq DNA Polymerase (Thermo-Fisher Scientific; Cat #12342010).
The PCR reaction was as follows: 95°C for 3.5 minutes; 30 cycles of 30 sec at 95.0°C, 30 sec at 50.0°C and
90 sec at 72.0°C; and finally 72.0°C for 10 minutes. PCR products were pooled, quantified, and visualized using
QIAxcel DNA Fast Analysis (Qiagen, Hilden, Germany). PCR products were normalized based on the concentration of DNA from 250-400
bp and pooled using the QIAgility liquid handling robot (Qiagen). Pooled PCR products were cleaned using the Gene Read Size
Selection kit (Qiagen; Cat #180514) according to the manufacturer’s protocol. Samples were sequenced on an
Illumina MiSeq using v3 chemistry (Illumina, San Diego, CA) according to the manufacturer’s instructions. PhiX
(5-20%) was added to the run to increase sample complexity. All sequence read data are available in the Sequence Read
Archive (BioProject ID PRJNA401190).

Adami A.J., Bracken S.J., Guernsey L.A., Rafti E., Maas K.R., Graf J., Matson A.P., Thrall R.S, & Schramm C.M. (2018). Early-Life Antibiotics Attenuate Regulatory T Cell Generation and Increase the Severity of Murine House Dust Mite-Induced Asthma. Pediatric research, 84(3), 426-434.

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Quantitative Transcriptional Analysis of Spheroids

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At each sampling timepoint, 16 spheroids were combined, and 3 independent experiments were performed (n = 3). RNA isolation was performed using either the RNeasy Plus Micro Kit (Qiagen), or the RNeasy Plus Mini Kit (Qiagen). The Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Fisher Scientific) was used for the reverse transcription using 1500 ng of the isolated RNA, as described in the manufacturer’s protocol. Primer3 software was used to design gene-specific primers (Supplementary Table S2) and GAPDH was used as a reference gene. 5ng cDNA template was used for each qPCR reaction in addition to 50% SYBR Green JumpStart Taq ReadyMix and 400 nM of each primer, to a final volume of 15 µL. The QIAgility liquid handling robot and Rotor-Gene Q cycler (Qiagen) were used for experimental setup and qPCR reaction, respectively. The qPCR cycling protocol included 3 min at 94°C for denaturation and a subsequent 40 cycles of 5 s at 95°C, 15s at 60°C, and 30 s at 72°C. Primer specificity was confirmed via melting curve analysis. Fetal Brain RNA (Takara Bio, Cat# 636526) and human cortical RNA (Takara Bio, Cat# 636561) were used for normalization. The ddCT method (Livak and Schmittgen, 2001 (link)) was used to analyze data obtained from three technical replicates for each gene.

Horánszky A., Shashikadze B., Elkhateib R., Lombardo S.D., Lamberto F., Zana M., Menche J., Fröhlich T, & Dinnyés A. (2023). Proteomics and disease network associations evaluation of environmentally relevant Bisphenol A concentrations in a human 3D neural stem cell model. Frontiers in Cell and Developmental Biology, 11, 1236243.

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Amplicon Library Preparation and Sequencing

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miRNA Quantification by RT-qPCR

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Real-time quantitative PCR (RT-qPCR) validation analysis was performed using the StepOnePlus^™ Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) and the miScript PCR System (Qiagen) according to the manufacturer’s instructions. For miRNA abundance level detection, 250 ng of the total RNA were converted into complementary DNA (cDNA). The resulting cDNA was then diluted to have 0.5 ng/μL input material for miRNA detection. All RT-qPCR experiments were carried out using the Liquid Handling Robot QIAgility^™ (Qiagen) before performing RT-qPCR. All primer assays used in the current study were provided by Qiagen. Moreover, miRNA reverse transcription control (miRTC) (Qiagen) was performed to assess the performance of the reverse transcription reaction. The melting curve analysis was used to control the specificity of RT-qPCR products. Specificity of amplicons was further confirmed by agarose gel electrophoresis.

Abu-Halima M., Meese E., Saleh M.A., Keller A., Abdul-Khaliq H, & Raedle-Hurst T. (2019). Micro-RNA 150-5p predicts overt heart failure in patients with univentricular hearts. PLoS ONE, 14(10), e0223606.

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Sperm miRNA Extraction and Analysis

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The miRNeasy Mini Kit (Qiagen) was used with a minor modification to extract total RNA including miRNA from sperm. Briefly, 200 µL of the purified sperm samples were homogenized in 700 µL QIAzol^® Lysis Reagent (Qiagen) mixed with Dithiothreitol (DTT) (80 mmol/L, Sigma) for 15 min. Subsequently, the procedure was completed according to the manufacturer’s instructions for Qiagen miRNeasy Mini Kit using QIAcube™ Robotic Workstation (Qiagen). DNase I treatment (Qiagen) was performed during the isolation to eliminate any contaminating genomic DNA. Total RNA concentration and purity were determined using Nanodrop-2000 (Thermo Fisher Scientific). Complementary DNA (cDNA) was generated in 20 µL reactions from 75 ng total RNA including miRNA using the miScript Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. The 5x miScript HiSpec Buffer was used to generate cDNA from total RNA including miRNA by reverse transcriptase using oligo-dT primers. After RT, the cDNA was diluted with miScript SYBR Green PCR Master Mix (Qiagen) and amplified to detect hsa-miR-19a-3p and hsa-miR-19a-3p. All RT-qPCR experiments were performed using the Liquid Handling Robot QIAgility™ (Qiagen) before performing RT-qPCR using the StepOnePlus™ Real-Time PCR system (Applied Biosystems). Moreover, RT negative controls and no template controls (NTC) were included.

Abu-Halima M., Becker L.S., Ayesh B.M, & Meese E. (2022). MicroRNA-targeting in male infertility: Sperm microRNA-19a/b-3p and its spermatogenesis related transcripts content in men with oligoasthenozoospermia. Frontiers in Cell and Developmental Biology, 10, 973849.

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Quantification of miR-126 Isoforms in Tourette Syndrome

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Using the RT-qPCR, the abundance levels of hsa-miR-126-3p and hsa-miR-126-5p were determined in 33 patients with TS and 33 age-matched HVs using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the TaqMan^® microRNA Assays (Thermo Fisher Scientific), according to the manufacturer’s recommendations. Briefly, complementary DNA (cDNA) was generated in 15-µL reactions by the reverse transcription (RT) of 75 ng total RNA using the TaqMan^® MicroRNA Reverse Transcription Kit and RT Primers Pool from the hsa-miR-126-3p (Assay ID: 002228) and hsa-miR-126-5p (Assay ID: 000451), along with the small nuclear RNA (snRNA) RNU6B (Assay ID: 001093). Following the RT step, qPCR was carried out using 1.0 µL of each TaqMan Primer Assay (20X) (a mixture of forward and reverse primers and a probe) (Thermo Fisher Scientific), 2.0 µL of RT reaction (1:5 diluted), 10.0 µL of 2XtaqMan™ Universal PCR Master Mix, no AmpErase™ UNG (Thermo Fisher Scientific), and 7.0 μL of DEPC-treated water to obtain a final volume of 20 μL. All RT-qPCR experiments were carried out in duplicates using the Liquid Handling Robot QIAgility™ (Qiagen) before performing qPCR according to the manufacturer’s recommendations.

Abu-Halima M., Oberhoffer F.S., Wagner V., Abd El Rahman M., Jung A.M., Zemlin M., Rohrer T.R., Meese E, & Abdul-Khaliq H. (2022). MicroRNA-126-3p/5p and Aortic Stiffness in Patients with Turner Syndrome. Children, 9(8), 1109.

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