Cells grown to confluence in 6-well plates were washed once with PBS. Cells were harvested and total RNA was isolated with the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. RNA concentrations were measured with a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). 1 μg of total RNA was reversely transcribed into cDNA with the Tetro reverse transcription kit (Bioline, Luckenwalde, Germany). Quantitative real-time PCR was carried out using SensiFAST SYBR Hi-ROX Mix (Bioline) on an ABI 7900HT cycler (Applied Biosystems, Darmstadt, Germany). HPRT1 was used as housekeeper gene. For analysis of relative changes, data was analyzed according to the ΔΔCT method.
Sensifast sybr hi rox mix
Manufactured by Meridian Bioscience
Sourced in United Kingdom, United States
The SensiFAST SYBR Hi-ROX Mix is a pre-mixed, ready-to-use solution for quantitative real-time PCR (qPCR) using SYBR Green detection. It contains all the necessary components for efficient and reliable qPCR, including a hot-start DNA polymerase, dNTPs, and SYBR Green I.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Absin (2 mentions)
Thermal cycler, by Bio-Rad (2 mentions)
Gotaq polymerase, by Promega (2 mentions)
Cfx connect 180 real time cycler, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Tetro reverse transcription kit, by Meridian Bioscience (1 mentions)
Abi 7900ht cycler, by Thermo Fisher Scientific (1 mentions)
High pure rna isolation kit, by Roche (1 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
96 well white plate, by Roche (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Chloroform, by Merck Group (1 mentions)
Revertaid reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
18 protocols using sensifast sybr hi rox mix
1
RNA Isolation and qPCR Analysis
2
Gene Expression Analysis in Synchronized C. elegans
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To assess gene expression levels, synchronized young adult worms were grown and frozen in liquid nitrogen. RNA extraction was performed according to a previously published method, with minor modifications [49 (link)]. Briefly, samples were collected and immediately snap frozen in liquid nitrogen. The following steps were performed after the samples were stored at −80 °C. Samples were resuspended in 500 μl of Trizol (catalog no. 10296010, Thermo Fisher). After 3 freeze–thaw cycles, 100 μl of chloroform (Merck, catalog no. C2432) was added. After centrifugation, the upper phase was collected and mixed with the same volume of 70% ethanol. The RNeasy Plus Mini Kit (Qiagen, catalog no. 74134) was used for further total RNA isolation according to the manufacturer’s instructions. Total RNA (500 ng) was used to generate cDNA using the SuperScript IV First-Strand Synthesis System (Invitrogen, catalog no. 18091050, Thermo Fisher). RT-qPCR was performed using SensiFAST SYBR Hi-ROX mix (2x; Bioline, catalog no. BIO-92020) in a LightCycler480 (Roche) with a 96-well white plate (Roche, catalog no. 4729692001). Fold changes in mRNA expression of the target genes were calculated using the ΔΔCt method. The mean expression levels of act-1 and cdc-42 were used as internal controls. The data are presented as mean ± SD (n = 3). The primers that were used in this study are shown in S5 Table .
3
Quantifying Gene Expression in Cells and Tissues
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Human cells and mice tissues were immersed in TRIzol® reagent (Life Technologies, Inc., Carlsbad, CA, USA) for the extraction of total RNA. All protocols followed manufacturer’s instructions with minor modifications. Total RNA ranging from 250 ng to 1 μg of total RNA was used to perform reverse transcription (RT) using the RevertAid™ Reverse transcriptase kit (Fermentas, Glen Burnie, Maryland, USA) according to manufacturer’s protocols. Real-time PCR reactions were performed using SensiFAST SYBR Hi-ROX mix (Bioline, Humber Road, London, UK), and the amplicons were detected and analyzed using the StepOne™ sequence detector (Life Technologies). The human gene expression was normalized to GAPDH expression. The mice gene expression level was normalized to Hprt1 expression. The relative expression was calculated by ΔΔCT methods.
4
Quantitative RT-PCR Gene Expression Analysis
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Total RNAs was extracted using a TruSeq RNA Library Prep Kit (Illumina). RNA was converted to cDNA using a reverse transcription reagent kit (Bioline, London, UK) and amplified using SensiFAST SYBR Hi ROX Mix (Bioline). ABI StepOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA) was used to conduct quantitative real-time polymerase chain reaction (qRT-PCR). 18S was the internal standard and relative gene expression was calculated using the 2-ΔΔCT method. CS1 was served as control values. Primers used for each gene were shown in S6 Table .
5
Real-Time PCR Analysis of Hematopoietic Transcription Factors
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Total RNA was extracted by using an illustra RNAspin Mini Isolation Kit (GE Healthcare). Isolated RNAs (4 μg) were subjected to cDNA synthesis with a RevertAid first strand cDNA synthesis kit (Fermentas). Real-time PCR was performed on an ABI StepOne Plus Real-Time PCR System (Applied Biosystems) using SYBR-Green reagents (SensiFAST SYBR Hi-ROX mix, Bioline). The samples were examined in triplicate for each assay and analyzed by using the comparative cycle threshold CT (∆∆CT) method [3 (link)]. The expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control for cDNA contents. Gene-specific amplification was conducted with the following primer sets: for EKLF, 5’-CGGCAAGAGCTACACCAAG-3’ (sense) and 5’-CCGTGTGTTTCCGGTAGTG-3’ (antisense); for GATA1, 5’-CAGTCTTTCAGGTGTACCC -3’ (sense) and 5’-GAGTGATGAAGGCAGTGCAG-3’ (antisense); for ALAS2, 5’-GCAGCACTCAACAGCAAG-3’ (sense) and 5’-ACAGGACGGCGACAGAAA-3’ (antisense); for PBGD, 5’-CGCCTCCCTCTAGTCTCTGCTTCT-3’ (sense) and 5’- GTTGCCACCACACTGTCCGTCTG-3’ (antisense); for GAPDH, 5’-TGGTATCGTGGAAGGACTCATGAC-3’ (sense) and 5’-ATGCCAGTGAGCTTCCCGTTCAGC-3’ (antisense).
6
RNA Isolation and qPCR Analysis
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iLNs or skins were dissected from E18.5 embryos and RNA isolated using the Qiagen RNeasy Plus Mini Kit (Qiagen). RNA was amplified using Ovation Pico WTA System V2 (Nugen). MEFs from chips and cells from co-culture experiments were collected using 0.05% trypsin-EDTA (Gibco) and RNA isolated using the Qiagen RNeasy Plus Micro or Mini Kit (Qiagen) and reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). We used StepOnePlus (Applied Biosystems) and SYBR Green PCR Master Mix (Applied Biosystems) or SensiFAST Sybr Hi-Rox Mix (Bioline) for qPCR analyses. Data were analyzed using the comparative Ct (ΔΔCt) method as described by the manufacturer. A list of primers used in this study is provided in Table S2.
7
Quantitative RNA Expression Analysis in BV2 Cells
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Total RNA was separated from BV2 cells using TRIzol reagent (Absin), and total RNA (1 μg) was reverse‐transcribed in a reaction mixture containing 1 U RNase inhibitor, 500 ng random primer, 3 mM MgCl2, 0.5 mM dNTP, 1X RT buffer, and 10 U reverse transcriptase (Promega). The cDNA served as a template for the PCR reaction, which was performed using Go Taq polymerase (Promega) and primers. The RT‐qPCR was carried out on the thermal cycler (Bio‐Rad) and on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using Sensi FAST SYBR Hi‐ROX Mix (Bioline). GAPDH was used as an endogenous reference for mRNA detection. Data were calculated using the 2−△△CT method.
31 (link) The experimental operating conditions were 95°C for 10 min, 95°C for 5 s, and 60°C for 60 s for 40 cycles. The primer sequences used in this study are listed in Table1 .
31 (link) The experimental operating conditions were 95°C for 10 min, 95°C for 5 s, and 60°C for 60 s for 40 cycles. The primer sequences used in this study are listed in Table
8
RNA Extraction and qPCR Analysis
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RNA was extracted by standard procedure with TRIzol reagent (Invitrogen). Equal amounts from each sample (500–800 ng) were used for cDNA syntheses applying the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific). Real‐time qPCR was performed on a CFX Connect™ 180 Real‐Time cycler (Biorad) (denaturation step: 95°C for 25 sec, annealing and elongation step: 60°C for 30 sec) using SensiFast SYBR™ Hi‐ROX mix (Bioline) with the following primers:
9
Pluripotency Marker Gene Expression
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An illustraTM RNAspin Mini (GE Healthcare, Little Chalfont, UK) was used to extract total RNA, and RNA was reverse-transcribed using SuperScript III RT (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacture’s specifications. For the real-time PCR, we used SensiFAST SYBR Hi-ROX Mix (Lot no: SFSH-717111A, Bioline, London, UK) and a StepOne Plus Real-Time PCR system (Applied Biosystems, Darmstadt, Germany) with specific primers. The results were calculated using the ∆∆CT equation and were expressed as multiples of change relative to a control sample. The primers used are as follows: GAPDH (forward: 5′-CAACTACATGGTTTACATGTTC-3′, reverse: 5′-GCCAGTGGACTCCACGAC-3′); Nanog (Forward: 5′-GTCCCGGTCAAGAAACAGAA-3′, reverse: 5′-TGCGTCACACCATTGCTATT-3′); Sox2 (forward: 5′-ATGGGTTCGGTGGTCAAGT-3′, reverse: 5′-ATGTGTGAGAGGGGCAGTGT-3′); Oct4 (forward: 5′-ATTCAGCCAAACGACCATCT-3′, reverse: 5′-ACACTCGGACCACATCCTTC-3′).
10
Comprehensive Gene Expression Profiling of Thymic Epithelial Cells
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Total RNA of enriched thymic epithelial cells, TEP1 and 1889c cells was isolated with the NucleoSpin RNAII kit (Macherey-Nagel). cDNA was prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). For qPCR analysis the StepOnePlus (Applied Biosystems) platform was used with SensiFAST SYBR Hi-ROX Mix (Bioline) as well as PikoReal™ Real-Time PCR System (Thermo Fisher Scientific) using Luminaris Color HiGreen qPCR Master Mix (Thermo Fisher Scientific) (for primer list see Table 1 ). Gene expression was normalized to β-actin, GUSB and HPRT1 housekeeping genes. Reverse transcription of 1889c RNA samples for miRNA analysis was completed with Megaplex™ RT Primers specific to human Pool A (Cat. No.: 4399966) and Pool B (Cat. No.: 4444281). MiRNA profiling was performed on Applied Biosystems Quantstudio™ 12K Flex Real-Time PCR System platform using TaqMan™ Array Human MicroRNA A (Applied Biosystems, Cat. No.: 4398965) and B Card (Applied Biosystems, Cat. No.: 4444910) containing 6 housekeeping genes (RNU44, RNU48, ath-miR159a and 4 U6 snRNAs) and 377 human miRNAs. Additionally 600 ng of total RNA was mixed with TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG (Applied Biosystems, Cat. No.: 4364103) for each array card. Gene expressions were analyzed using Expression Suite Software version 1.1.
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