HeLa cells were incubated in serum-free DMEM for 4 h with siRNA (10 nM) against HIF-1α (Qiagen) or Lpin1[6] (link) in the presence of Lipofectamine™ RNAiMAX (Invitrogen). AllStars siRNA (Qiagen) was used as negative control.
Hif 1α
Manufactured by Qiagen
Sourced in Netherlands
The HIF-1α is a lab equipment product manufactured by Qiagen. It is used to detect and measure the levels of the hypoxia-inducible factor 1-alpha (HIF-1α) in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Qiagen (2 mentions)
Hif 2α, by Qiagen (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Allstars sirna, by Qiagen (1 mentions)
Vegf a, by Qiagen (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex rt pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Glut1, by Qiagen (1 mentions)
Vascular endothelial growth factor (vegf), by Qiagen (1 mentions)
Human monocyte nucleofactor kit, by Lonza (1 mentions)
18s ribosomal rna, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Simpliamp thermal cycler, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
8 protocols using hif 1α
1
Silencing HIF-1α and Lpin1 in HeLa Cells
2
Quantitative PCR for HIF-1α Expression
3
Real-Time PCR for Transcription Factors
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Real‐time polymerase chain reaction (RT‐PCR) was performed using a TaqMan array with customized targets and a catalogued array for transcription factors (Applied Biosystems/Life Technologies, Lofer, Austria). RNA was pooled from two independent experiments after 1 day (HEL) or 2 days (CMK) of culture in ID (slower growth of CMK compared with HEL). Three independent 5‐day experiments of CD61+ MACS sorted MEGs were pooled. HPRT1 and 18S were used as endogenous controls, and cut‐offs were set at 2.0 for upregulation and 0.5 for downregulation. Individual genes such as TfR1, HIF2α, HIF1α, VEGFA (Qiagen, Hilden, Germany), VEGFR1, and VEGFR2 (VBC Biotech, Vienna, Austria) were verified using SYBR green (Applied Biosystems/Life Technologies). Analysis was performed using Applied Biosystems software and LinRegPCR 37 . Relative expression was calculated using 2 − ( C T Targe t − , C T Housekeeping ) 2 − ( Δ C T ID − Δ C T IR )
4
Gene Expression Analysis in Hypoxia
5
Silencing HIF-1α and HIF-2α in CB-MNCs
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Freshly isolated CB-MNCs were transfected with dssiRNA HIF-1α and HIF-2α (Qiagen, Venlo, Netherlands) using ‘Human monocyte nucleofactor kit' (Lonza, VPA-1007). Briefly, for each condition an equal amount of CB-MNCs was centrifuged, and 100 μl nucleofactor was added to the cell pellet. From both dssiRNA HIF-1α and HIF- 2α, 1 μg was added to the cell suspension, and transferred to the cuvette and placed in the electroporation system (Amaxa, Lonza Verviers) according to the manufacturer. Transfected CB-MNCs were resuspended in complete EGM medium, transferred to 0.1% gelatin coated wells, and further cultured under 1% O2 according the CB-ECFC culture protocol. Mock transfected cells (only electroporation step, no dssiRNA transfection) served as a control in 20 and 1% O2. Further details are given in Nauta et al. (30 (link)).
6
Inflammatory Cytokine Expression Profiling
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Total RNA was extracted from islets using TRIzol (Thermo Fischer Scientific, Waltham, MA, USA) and was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Thermo Fischer Scientific). Template cDNA was mixed with SYBR® Green PCR Master Mix (Thermo Fischer Scientific) and primers of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-18, chemokine C-C motif ligand 2 (CCL2), C-X-C motif ligand 8 (CXCL8), high-mobility group box protein 1 (HMGB-1), tissue factor (TF) (Invitrogen, Carlsbad, CA, USA), or hypoxia-inducible factor 1-alpha (HIF-1α) (Qiagen Inc., Valencia, CA, USA). The reverse transcription PCR was run using SimpliAmp™ Thermal Cycler (Thermo Fischer Scientific) with the following program: 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 60 s. Quantitative analysis was performed with 18S ribosomal RNA (Thermo Fischer Scientific) as an internal control, and relative expression levels were calculated with the 2−ΔΔCt method. All measuring samples were triplicated.
7
Quantitative PCR for HIF-1α Expression
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Q-PCR was performed as previously described.10 (link) Primer sets for HIF-1α (cat no. QT01039542) and β-actin (cat no. QT01680476) were purchased from Qiagen (Limburg, Netherlands) who retain the right to withhold primer sequence information. Fold change was calculated using the ddCT (delta-delta-Ct) method, standardised to β-actin. A dissociation step was performed to verify a single product was produced with each primer set. The size of PCR products was also verified by resolution on a 2% polyacrylamide gel with both HIF-1α and β-actin giving an approximate amplicon length of 104 bp. All Q-PCR primer assays in this study were at least 96% efficient.
8
Hypoxia Signaling Pathway Regulation
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U2OS cells were transfected with control siRNA (10 nM; 5′- UUCUCCGAACGUGUCACGU -3′) or siRNA against Arnt (5′-GGAACAAGAUGACAGCCUATT -3′), Miz-1 (5′- GCCUCAUCAGCCUGCUGAATT -3′), Hif1α (5′- GAAGAACUAUGAACAUAAATT -3′) and Hif2α (5′- CGGAUAGACUUAUUGCCAATT -3′) (Qiagen) using the HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions. The cells were cultured for 48 hours and then placed at 21% or 1% O2 for 6 hours before harvested.
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