Total RNA was extracted using the 6100 Nucleic Acid Prepstation (Applied Biosystems, Darmstadt, Germany) following the manufacturer's protocol. In all, 150 ng total RNA was reverse transcribed with the ThermoScript RT-PCR System for First-Strand cDNA Synthesis (Invitrogen, Darmstadt, Germany), SuperScript III Reverse Transcriptase (Invitrogen) or Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. Quantitative RT-PCR was performed in ABI Prism 7000 Sequence Detection System (Applied Biosystems) using the SensiMix SYBR Kit (Bioline, Luckenwalde, Germany) or Taqman Gene Expression Assay (Invitrogen). The mRNA levels were normalized to human L28 mRNA. Following primers and assays were used for quantitative RT-PCR: human AM forward, 5′-GGA TGC CGC CCG CAT CCG AG-3′ human AM reverse, 3′-GAC ACC AGA GTC CGA CCC GG-5′ human L28 forward, 5′-ATG GTC GTG CGG AAC TGC T-3′ human L28 reverse, 5′-TTG TAG CGG AAG GAA TTG CG-3′ Taqman Gene Expression Assay for HIF-1α (Hs00936368_m1).
6100 nucleic acid prepstation
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The 6100 Nucleic Acid Prepstation is a laboratory instrument designed for the automated extraction and purification of nucleic acids, such as DNA and RNA, from a variety of sample types. The core function of the instrument is to perform the necessary steps to isolate and concentrate nucleic acids, preparing them for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Sensimix sybr kit, by Meridian Bioscience (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Tissuelyser, by Qiagen (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Thermoscript rt pcr system for first strand cdna synthesis, by Thermo Fisher Scientific (1 mentions)
Transcriptor high fidelity cdna synthesis kit, by Roche (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Thermal cycler, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
7 protocols using 6100 nucleic acid prepstation
1
Transcriptome Analysis of AM and HIF-1α
2
Total RNA Extraction from Skin
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RNA was prepared according to a standard phenol‐chloroform total RNA extraction method where skin samples were homogenized in QIAzol® Lysis Reagent (QIAGEN Sciences, Maryland 20847, USA), added chloroform, vortexed and centrifuged for phase separation. The RNA containing phase was ethanol precipitated and purified on an Applied Biosystems 6100 Nucleic Acid Prep Station. RNA integrity was measured on the Fragment Analyzer platform (AATI, IA, USA) using standard sensitivity RNA analysis kit.
3
Whole Blood RNA Isolation Protocol
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The whole blood was defrosted. One volume of whole blood was resuspended in 1 volume of PBS and in 2 volumes of Purification Lysis Solution (Applied Biosystems, Foster City, CA). RNA was isolated using 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol, and then stored at −80°C.
The RNA concentration and purity were assessed spectrophotometrically by measuring their absorbance at 260 nm and 280 nm. RNA fragmentation state was evaluated by 1.5% agarose gel.
The RNA concentration and purity were assessed spectrophotometrically by measuring their absorbance at 260 nm and 280 nm. RNA fragmentation state was evaluated by 1.5% agarose gel.
4
Liver Gene Expression in Work Cycle
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Following the third work session, animals were fasted for 2 h to avoid the immediate effects of food intake on gene expression, anaesthetized with isoflurane, and sacrificed by decapitation. AW were sacrificed at ZT0, before the transition from dark to light phase, and RW at ZT12, before the transition from light to dark phase. A separate group of undisturbed animals never exposed to simulated work were used as time-matched controls, and sacrificed at the same zeitgeber times as experimental animals (AW control: ZT0; and RW control: ZT12). Liver tissue was harvested, flash frozen, and stored at −80 °C until analysis. Samples were homogenized using a TissueLyser (Qiagen, Valencia, CA, USA). RNA extraction was performed using a 6100 Nucleic acid PrepStation (Applied Biosystems, Foster City, CA, USA). A total of 20 ng RNA was transcribed to cDNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems). Real-time polymerase chain reaction (PCR) was run on the Applied Biosystems 7900 Real-Time PCR System, with each sample run in triplicate. Relative gene expression levels were determined using the comparative ΔCt method, using β-actin (Actb) and ribosomal protein lateral stalk subunit P0 (Rplp0) as endogenous controls. Sequence names, main function, accession numbers, and primer sequences are shown in Table 1 .
5
Gene Expression Analysis of Mouse Tumors
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Snap-frozen tumors were disrupted using 6 mm stainless steel beads in the TissueLyser (Qiagen, Hilden, Germany). RNA was extracted using the 6100 Nucleic acid prepstation (Applied Biosystems, California, USA), according to the manufacturer’s instructions. copyDNA was synthesized using random primers in a thermal cycler (Applied Biosystems). Primers specific for mouse hypoxanthine phosphoribosyltransferase 1 (HPRT1), programmed death-ligand 1 (PD-L1), and interferon-γ (IFN-γ) were used and relative gene expression was determined using the iQ SYBR Green supermix-CFX Connect Real-Time System (BioRad Laboratories, Temse, Belgium). The comparative threshold cycle method was used to calculate gene expression normalized to HPRT1 as a reference gene.
6
Whole Blood RNA Extraction Procedure
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Whole blood was sampled into Tempus Blood RNA tubes at time points 0, 2h, 4h, and 6h, and RNA extraction from leukocytes performed on the 6100 Nucleic Acid PrepStation (Applied Biosystems, USA). Amount and quality of the extracted RNA was verified by the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). All RNA samples were qualitatively adequate with RNA integrity numbers between 6.9 and 9.7.
7
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using the 6100 Nucleic Acid Prepstation (Applied Biosystems, Darmstadt, Germany) following the manufacturer's instruction. Total RNA (200–500 ng) was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen, Darmstadt, Germany) or SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Quantitative RT-PCR was performed in ABI Prism 7000 Sequence Detection System (Applied Biosystems) using SensiMix SYBR Kit (Bioline, Luckenwalde, Germany) or Taqman Gene Expression Assay (Applied Biosystems). The mRNA levels were normalized to human L28 mRNA. The following primers and assays were used: human GLUT1 forward, 5′-GGC CTT TTC GTT AAC CGC TT-3′ human GLUT1 reverse, 5′-AGC ATC TCA AAG GAC TTG CCC-3′ human L28 forward, 5′-ATG GTC GTG CGG AAC TGC T-3′ human L28 reverse, 5′-TTG TAG CGG AAG GAA TTG CG-3′ human VEGF-A forward, 5′-GCA GAA TCA TCA CGA AGT GG-3′ human VEGF-A reverse, 5′-GCA TGG TGA TGT TGG ACT CC-3′ Taqman Gene Expression Assay for HIF-1α (Hs00936368_m1), HIF-2α (Hs01026149_m1), BNIP3 (Hs00969293_mH), p50 (Hs00765730_m1).
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