When Mosquito HV bites Biomark HD: An automated workflow for high-throughput qPCR

Manjunath, Harshitha Shobha; Kalikiri, Mahesh Kumar Reddy; Kabeer, Basirudeen Syed Ahamed; Tomei, Sara

doi:10.1016/j.slast.2021.12.007

Cited by 3 publications

(5 citation statements)

References 17 publications

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“…Automated solutions are currently applied in many fields of life sciences; especially in genomics, laboratory specialists are streamlining their protocols by using automated workstations 26 – 28 . Automated solutions help cutting costs associated to manual labor and also help wet-lab specialists who would not have to spend long time processing samples 15 . In our study we questioned whether consistent expression profiles could be obtained from the automated isolation of volumes of blood < 50 μl.…”

Section: Discussionmentioning

confidence: 99%

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Technical assessment of different extraction methods and transcriptome profiling of RNA isolated from small volumes of blood

Kalikiri

Manjunath

Vempalli

et al. 2023

Sci Rep

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Transcriptome profiling of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Recently, finger-stick blood collection systems have allowed a less invasive and quicker collection of peripheral blood. Such non-invasive sampling of small volumes of blood offers practical advantages. The quality of gene expression data is strictly dependent on the steps used for the sample collection, extraction, preparation and sequencing. Here we have: (i) compared the manual and automated RNA extraction of small volumes of blood using the Tempus Spin RNA isolation kit and the MagMAX for Stabilized Blood RNA Isolation kit , respectively; and (ii) assessed the effect of TURBO DNA Free treatment on the transcriptomic data of RNA isolated from small volumes of blood. We have used the QuantSeq 3′ FWD mRNA-Seq Library Prep kit to prepare RNA-seq libraries, which were sequenced on the Illumina NextSeq 500 system. The samples isolated manually displayed a higher variability in the transcriptomic data as compared to the other samples. The TURBO DNA Free treatment affected the RNA samples negatively, decreasing the RNA yield and reducing the quality and reproducibility of the transcriptomic data. We conclude that automated extraction systems should be preferred over manual extraction systems for data consistency, and that the TURBO DNA Free treatment should be avoided when working on RNA samples isolated manually from small volumes of blood.

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Section: Discussionmentioning

confidence: 99%

“…However, there currently exist many liquid-handling workstations on the market, each one of them offers different degrees of flexibility. Hamilton Robotics (Hamilton, NV, USA), for instance, offers autonomous programming 15 . In this study the Hamilton NGS Star platform has been employed for automated RNA extraction.…”

Section: Introductionmentioning

confidence: 99%

Technical assessment of different extraction methods and transcriptome profiling of RNA isolated from small volumes of blood

Kalikiri

Manjunath

Vempalli

et al. 2023

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…Automated solutions are currently applied in many elds of life sciences; especially, in genomics, laboratory specialists are streamlining their protocols by using automated workstations [26][27][28] . Automated solutions help cutting costs associated to manual labor and also help wetlab specialists who would not have to spend long time processing samples 15 . In our study we questioned whether consistent expression pro les could be obtained from the automated isolation of volumes of blood < 50 𝜇l.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, technical improvements are required to make the gene expression pro ling of small volumes of blood a reliable and reproducible technique 12 . Automated work ows offer several advantages for large-scale projects, as they increase sample throughput and reduce cost and manual errors [13][14][15][16] . The MagMAX for Stabilized Blood RNA Isolation Kit (ThermoFisher Scienti c) employs a magnetic bead-based technology to purify RNA from blood stored in Tempus solution.…”

Section: Introductionmentioning

confidence: 99%

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Transcriptome sequencing of RNA isolated from small volumes of blood stabilized in Tempus solution: a technical assessment of different extraction methods and DNase treatment

Kalikiri

Manjunath

Vempalli

et al. 2022

Preprint

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Transcriptome profiling of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. RNA sequencing technologies offer many advantages for transcriptomic profiling over other technologies, including the ability to detect novel transcripts, a wide dynamic range of transcript detection, high specificity and sensitivity and the ability to detect low-abundance transcripts. Recently, finger-stick blood collection systems have allowed a less invasive and quicker collection of peripheral blood that does not necessarily require medical infrastructures. Such non-invasive sampling of small volumes of blood offers practical advantages, allowing large-scale projects. The quality of gene expression data is strictly dependent on the steps used for the sample collection, extraction, preparation and sequencing. Here we have: i. compared the manual and automated RNA extraction of small volumes of blood using the Tempus Spin RNA isolation kit (ThermoFisher Scientific, USA) and the MagMAX™ for Stabilized Blood RNA Isolation Kit (ThermoFisher Scientific, USA), respectively; and ii. assessed the effect of TURBO DNA Free treatment on the transcriptomic data of RNA isolated from small volumes of blood. RNA Libraries were prepped using the QuantSeq 3' FWD mRNA-Seq Library Prep Kit (Lexogen, Austria). Library QC was performed on the LabChip GXII. Libraries were quantified using KAPA Library quantification kit by qPCR on the LightCycler 480 II (Roche Diagnostics, Basel, Switzerland). Libraries were pooled on the Hamilton MicroLab Star (Hamilton, Reno, NV, USA ) and sequenced on the Illumina NextSeq 500 system. The QC of the sequencing data was performed as recommended by Illumina. Reads were mapped to the human genome GRCh38.p13 (Genome Reference Consortium Human Build 38), INSDC Assembly GCA_000001405.28, Dec 2013) using STAR_2.6.1d aligner, and featureCounts v2.0.0 was used to generate the raw counts. We used DESeq2 (v1.32.0) to normalize read counts with standard settings. Normalized data was transformed using variance-stabilizing transform (VST) and removed of the batch effect using limma::removeBatchEffect from Lima package (v3.48.2). Heatmaps, correlation matrices and PCA plots were generated as relevant. Transcriptomic profiles were overall consistent, however the samples isolated manually displayed a higher variability in the transcriptomic data as compared to the other samples. The TURBO DNA Free treatment affected the RNA samples negatively, decreasing the RNA yield and reducing the quality and reproducibility of the transcriptomic data. We conclude that automated extraction systems should be preferred over manual extraction systems for data consistency, and that the TURBO DNA Free treatment should be avoided when working on RNA samples isolated manually from small volumes of blood.

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Automation and miniaturization of high-throughput qPCR for gene expression profiling

Raveendran,

Saeed,

Kalikiri

et al. 2024

SLAS Technology

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When Mosquito HV bites Biomark HD: An automated workflow for high-throughput qPCR

Cited by 3 publications

References 17 publications

Technical assessment of different extraction methods and transcriptome profiling of RNA isolated from small volumes of blood

Technical assessment of different extraction methods and transcriptome profiling of RNA isolated from small volumes of blood

Transcriptome sequencing of RNA isolated from small volumes of blood stabilized in Tempus solution: a technical assessment of different extraction methods and DNase treatment

Automation and miniaturization of high-throughput qPCR for gene expression profiling

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