FC and BG tissue were processed using the Sample Processing Kit (Affymetrix, #QS0106, Santa Clara, CA, USA) prior to gene quantification with the QuantiGene Plex Assay Kit (Affymetrix, #QP1014), both according to manufacturer’s instructions. The QuantiGene Plex assay is a multiplex assay to quantify mRNA expression directly from lysed tissue samples. Powdered brain tissue was weighed and treated with 300 μl of working tissue homogenate solution (Tissue homogenizing solution and proteinase K at a ratio of 60:1) per 10 mg of tissue. The samples were diluted to 16x to ensure fluorescent intensity readings were in the linear range. The tissue weight, ratio of tissue homogenizing solution to proteinase K, and sample dilutions were determined in previous optimization experiments. All incubation steps were performed in the Vortemp56 (Labnet, Edison, NJ, USA). Bead fluorescence was read on the Luminex 100/200 plate reader (Luminex Corporation, Austin, TX, USA) and Luminex xPonent for LX100/LX200 software (Version 3.1, Build 971). Fluorescence readings for each gene were normalized to housekeeping gene (RPS13) fluorescence levels and expressed as fold change compared to SIV− controls.
Quantigene plex assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantiGene Plex Assay kit is a multiplex gene expression profiling solution that allows for the simultaneous measurement of multiple target genes from a single sample. The kit utilizes a branched DNA signal amplification technology to provide quantitative and sensitive detection of gene expression levels.
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Lab products found in correlation
Quantigene sample processing kit, by Thermo Fisher Scientific (3 mentions)
Luminex flexmap 3d system, by Thermo Fisher Scientific (2 mentions)
Quantigene plex panel, by Thermo Fisher Scientific (2 mentions)
Quantigene sample processing kit for cultured cells, by Thermo Fisher Scientific (2 mentions)
Vortemp 56, by Labnet (1 mentions)
7 protocols using quantigene plex assay kit
1
Quantifying mRNA expression from tissue samples
2
Ferret Ghrelin mRNA Quantification
3
Tracheal Wnt Signaling Expression
4
Quantitative Analysis of SLC14A1 in Bladder Cancer
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Data mining is described in the Supplementary materials . For the QuantiGene branched DNA assay and immunohistochemistry, the Institutional Review Board of Chi Mei Medical Center approved the retrospective retrieval (IRB10302015) of 42 and another 340 primary UTUCs, as well as 36 and another 295 UBUCs with available tissue blocks from patients who underwent surgical treatment with curative intent between 1996 and 2004, while samples from those who underwent palliative resection were excluded (Supplementary materials ). To determine the clinical relevance of the SCL14A1 transcript level, 36 UBUCs and 42 UTUCs with normal, pTa-pT1 and pT2-pT4 specimens were evaluated. For immunohistochemistry, another 340 primary UTUCs and 295 UBUCs with available tissue blocks were used (Supplementary materials ). One specific probe targeting the SLC14A1 transcript was designed for QuantiGene™ Sample Processing Kit, formalin-fixed paraffin-embedded (FFPE) samples (QS0107, ThermoFisher, USA) and QuantiGene™ Plex Assay Kit (QP1013, ThermoFisher) based on the user guides (Supplementary materials ). Immunohistochemical (IHC) staining was performed on representative tissue sections cut from FFPE tissues at 4-µm thickness as in our previous study 14 (link) by probing specific anti-human antibodies (Supplementary materials ).
5
Quantifying Ribosomal Protein mRNA Levels
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Cells were treated with 0.1% DMSO or varying concentrations of C6 or C16 for 24 hr. A custom QuantiGene Plex panel (Thermo Fisher Scientific) was used in conjunction with the QuantiGene Sample Processing Kit for cultured cells (Thermo Fisher Scientific), and QuantiGene Plex Assay kit (Thermo Fisher Scientific) to quantify transcripts following the manufacturer’s instructions. Probe regions and accession numbers are as follows: RPS24 (NM_001026, region 5-334), RPL35 (NM_007209, region 2-430), RPL26 (NM_000987, region 37-445), RPS14 (NM_005617, region 61-552), RPL32 (NM_000994, region 95-677), RPS11 (NM_001015, region 139-634), RPL14 (NM_003973, region 108-530), and GAPDH (NM_002046, region 2-407). The average net mean fluorescence intensity was read on a Luminex FLEXMAP 3D System (Invitrogen). Signals from RPGs were normalized internally to those from GAPDH, and then to the DMSO control. Dose–response curves from the mean of biological replicates were calculated with the R package drc (Ritz et al., 2015 (link)).
6
TIGIT+ and TIGIT- GC-Tfh Cell Analysis
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TIGIT+ and TIGIT- GC-Tfh cells were sorted by FACSAria. The sorted cells were directly collected into the lysis mixture and prepared the cell lysates by using QuantiGene Sample Processing Kit (Thermo Fisher Scientific). The expression levels of mRNA were analyzed with using QuantiGene Plex Assay kit (Thermo Fisher Scientific) and MAGPIX system (Luminex Corporation, Austin, Texas) according to the manufactures’ protocols. Fold changes to average of three house-keeping genes were calculated after subtraction of background.
7
QuantiGene Plex Assay for Transcripts
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Cells were treated with 0.1% DMSO or varying concentrations of C6 or C16 for 24 hours. A custom QuantiGene Plex panel (ThermoFisher Scientific) was used in conjunction with the QuantiGene Sample Processing Kit for cultured cells (ThermoFisher Scientific), and QuantiGene Plex Assay kit (ThermoFisher Scientific) to quantify transcripts following the manufacturer’s instructions. Probe regions and accession numbers are as follows: RPS24 (NM_001026, region 5-334), RPL35 (NM_007209, region 2-430), RPL26 (NM_000987, region 37-445), RPS14 (NM_005617, region 61-552), RPL32 (NM_000994, region 95-677), RPS11 (NM_001015, region 139-634), RPL14 (NM_003973, region 108-530), and GAPDH (NM_002046, region 2-407). The Average Net Mean Fluorescence Intensity was read on a Luminex FLEXMAP 3D System (Invitrogen). Signals from RPGs were normalized internally to those from GAPDH, and then to the DMSO control. Dose response curves from the mean of biological replicates were calculated with the R package drc (Ritz et al, 2015 (link)).
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