The strains were grown for 24 h in YNB medium with glycerol (100 g/L). Consequently, cultures were centrifuged for 5 min at 12,000g. RNA was isolated using Total RNA Mini Plus kit (A&A Biotechnology, Poland) followed by DNase I (Thermo Scientific, USA) treatment according to the producer’s instructions. RNA quantities were measured using a Biochrom WPA Biowave II spectrophotometer (Biochrom Ltd., UK) equipped with a TrayCell (Hellma Analytics, Germany), next the isolated RNAs were stored in a − 80 °C freezer. cDNA synthesis was proceeded using Maxima First Strand cDNA Synthesis kits for RT-qPCR (Thermo Fisher Scientific). qRT-PCR analyses were performed using the DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific) using the Eco Real-Time PCR System (Illumina, USA). The primers qVHb-F (5′-ACCAGCAGACCATCAACATC-3′) and qVHb-R (5′-GCCCATGTCGAATAAAGGTC-3′) bind to the codon-optimized VHb gene, resulting in a 131 bp product. The genes expression level was normalized to the actin gene (ACT-F 5′-GAGTCACCGGTATCGTTC-3, ACT-R 5′-GCGGAGTTGGTGAAAGAG-3′) and analyzed using the ddCT method [31 (link)]. Samples were analyzed in three repetitions.
Dynamo flash sybr green qpcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland, Lithuania, United Kingdom
The DyNAmo Flash SYBR Green qPCR Kit is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains the necessary components, including a SYBR Green I-based hot-start DNA polymerase, for the amplification and detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (27 mentions)
Rneasy mini kit, by Qiagen (11 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (11 mentions)
Quantitect reverse transcription kit, by Qiagen (10 mentions)
Dynamo cdna synthesis kit, by Thermo Fisher Scientific (9 mentions)
Dnase 1, by Thermo Fisher Scientific (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Eco real time pcr system, by Illumina (5 mentions)
Total rna mini plus kit, by A&A Biotechnology (5 mentions)
Traycell, by Hellma (5 mentions)
Biochrom wpa biowave 2 spectrophotometer, by Harvard Bioscience (5 mentions)
Cfx96 qpcr system, by Bio-Rad (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (4 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Abi 7500, by Thermo Fisher Scientific (3 mentions)
Lightcycler 480, by Roche (3 mentions)
Maxima first strand cdna synthesis kit for rt qpcr, by Thermo Fisher Scientific (3 mentions)
128 protocols using dynamo flash sybr green qpcr kit
1
Quantitative Analysis of VHb Gene Expression
2
Gene Expression Analysis of Treated Cells
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Total RNA from treated and control cells were isolated after 24, 48, and 72 h using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For each sample, 2 μg of total RNA was reversely transcribed using the QuantiTect Reverse Transcription Kit (Qiagen Inc., Valencia, CA, USA). Gene expression was determined using the DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific Inc., Foster, CA, USA) on the Stratagene Thermocycler (Mx3000). The primers used are listed in Table 1 .
3
BAP1 Mutation Impacts on Stress Response
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Effect of BAP1 mutation on up-regulation of Hsp70, Hsp90 and transactivation of three target genes (MCM3, TP53I3 and CDKN1B) at cellular level was determined by RT-qPCR. HEK293T cells were transfected with empty vector (EV), wild type and mutant plasmids. After 48 h, total RNA was isolated using RNeasy mini kit (QIAGEN). Reverse transcription was performed using Verso cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s protocol. Quantitative real-time PCR was determined by using Dynamo Flash SYBR Green qPCR kit (Thermo Scientific) on a C1000 Touch Thermal cycler real-time machine (Bio-Rad) (Supplementary Table 1 ).
4
Real-Time PCR Gene Expression Analysis
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The Real-Time PCR was performed by the Applied Biosystems 7500/7500 Fast Real-time System (ABI, USA) following the manufacturer’s protocol of DyNAmo Flash SYBR Green qPCR Kit (Thermo scientific, USA). The reaction was run in a 10 μl volume containing 20 ng of cDNA, 0.3 μM of each primer and 5 μl SYBR green Master Mix. The PCR parameters were 95°C for 2 min, followed by 38 cycles of 95°C for 5 s, 58°C for 20 s and 72°C for 20 s. The specific primers were listed in Table 1
5
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from cells using an RNA Simple Total RNA isolation kit (Promega RNA Tissue/Cell Miniprep System) according to the manufacturer’s instructions. In-column DNase digestion was performed to obtain DNase-free RNA. RNA was quantified using Nanodrop (Thermo Fisher Scientific, Inc., Washington, DC, USA), and agarose gel electrophoresis was performed to assess RNA integrity. RNA samples were then reverse transcribed into cDNA using a Verso cDNA synthesis kit (AB1453A, Thermo Fisher Scientific, Inc., Washington, DC, USA). RT-qPCR was performed using a DyNAmo Flash SYBR Green qPCR kit (F415S, Thermo Fisher Scientific, Inc., Washington, DC, USA). The reaction cycles were as follows: incubation at 95 °C for 7 min, 40 cycles at 95 °C for 15 s and 60 °C for 20 s. The PCR products were subjected to a melting curve analysis to confirm amplification specificity. mRNA levels were then normalized with respect to mRNA levels of GAPDH using the 2−ΔΔCq method [37 (link)]. PCR primer sequences are provided in Table S1 .
6
Validating RNA-seq Results via qRT-PCR
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The RNA preparations were extracted in triplicate with three biological replications, as described above. Twenty genes were randomly selected for the validation of the RNA-seq results using qRT-PCR. All of the gene-specific primers were designed by the Primer 5.0 program (Table S1 ), and th qRT-PCR reactions were performed using a DyNAmo Flash SYBR Green qPCR kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the CFX96 qPCR System (Bio-Rad Hercules, CA, USA). The expression levels were calculated by the 2−ΔΔCt method, and were normalized to that of the reference gene CsaTIP41 [50 (link)].
7
ITPR Expression and Knockdown in Cells
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RNA was extracted by NucleoSpin® RNA extraction kit (Macherey‐Nagel #740955) and reverse transcribed by Maxima first strand cDNA synthesis kit (Thermo Fischer). RT‐qPCR was performed using DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) with specific primers for ITPR1, ITPR2, ITPR3, and GAPDH (Table S2 ) using Bio‐Rad CFX Maestro 1.1 software (Bio‐Rad). ITPR3 siRNA knockdown experiments were conducted as described19 using ITPR3 ON‐TARGETplus SMARTpool siRNA (Dharmacon #L‐006209‐00‐0005) and ON‐TARGETplus Non‐targeting siRNA (Dharmacon #D‐001810‐01‐05).
8
FOSL1 Knockdown Modulates Diabetic Progression
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Short hairpin (sh) RNA targeting FOSL1 (sh-FOSL1), sh-negative control (sh-NC), adenovirus vector (Ad)-sh-FOSL1, and Ad-sh-NC were purchased from Sangon Biotech (Shanghai, China). Streptozotocin (STZ) was obtained from Sigma Aldrich (San Luis, MO, USA). The RevertAid H Minus First Strand cDNA Synthesis kit, DyNAmo Flash SYBR Green qPCR kit, and apoptosis detection kit were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies (FOSL1, p-ERK, ERK, c-fos, c-jun, and GAPDH) and the HRP-conjugated secondary antibody used for western blotting were procured from Abcam (Cambridge, UK).
9
NUDT21 mRNA Expression Analysis
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RNA sample isolation was conducted utilizing the PureLink™ RNA Mini Kit, and cDNA was generated using the DyNAmo cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). For NUDT21 mRNA detection, DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific, Waltham, MA, USA) was applied to qRT-PCRs on Light Cycler 480 II Real-Time PCR System (Roche Diagnostics, Basel, Switzerland) using GAPDH as the control. The primer sequences of NUDT21 [16 (link)] are presented in Table 1 . The relative expression was calculated using the 2−ΔΔCT method.
10
Total RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from the placental villous tissues, explants, and cells using an RNA Simple Total RNA kit (Promega RNA Tissue/Cell Miniprep System) according to the manufacturer's instructions. RNA was quantified using Nanodrop (Thermo Fisher Scientific, Inc.), and agarose gel electrophoresis was performed to check the integrity of the isolated RNA. The isolated RNA was reverse transcribed into cDNA using the Verso cDNA synthesis kit (AB1453A, Thermo Fisher Scientific, Inc.). RT-qPCR was performed using DyNAmo Flash SYBR Green qPCR kit (F415S, Thermo Fisher Scientific, Inc.). The RT-qPCR reaction cycles were as follows: incubation at 95 °C for 7 min, 40 cycles at 95 °C for 15 s, and 60 °C for 20 s. The PCR products were subjected to a melting curve analysis to confirm the amplification specificity. Levels of mRNA were normalized with respect to GAPDH mRNA levels and evaluated using the 2-ΔΔCq method10 (link). PCR primer sequences have been provided in the Suppl. Table 1 .
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