Total RNA was extracted from the cortex of the ischemic hemisphere using RNApure Tissue Kit (CWbio Co., Ltd., Beijing, China) according to the manufacturer's protocol. The quantity of total RNA was measured using Nano-Drop 2000 (Thermo, MA, USA). Reverse transcription was performed using SuperRT cDNA Kit (CWbio Co., Ltd.). Quantitative RT-PCR was carried out on a ABI 7500 real-time fluorescence quantitative PCR apparatus (Applied Biosystems, Foster City, CA, USA) using a UltraSYBR Mixture (With ROX) kit (CWbio Co., Ltd.). Amplification procedure was as follows: 95°C for 10 minutes, 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The oligonucleotide primer sequences were designed as follows: HO-1 (92 bp), forward: TCA CTG GCA GGA AAT CAT CC, reverse: CTG AGA GGT CAC CCA GGT A; GADPH (138 bp), forward: TGG AGT CTA CTG GCG TCT T, reverse: TGT CAT ATT TCT CGT GGT TCA. All samples were assayed in triplicate, and relative gene expression was quantified using the 2-ΔΔCt method as previously described (Livak and Schmittgen, 2001).
Ultrasybr mixture with rox kit
Manufactured by CWBIO
Sourced in China
The UltraSYBR Mixture (with ROX) Kit is a real-time PCR (qPCR) reagent designed for efficient and sensitive DNA amplification and detection. The kit contains a ready-to-use master mix that includes the SYBR Green I dye, a passive reference dye (ROX), and necessary buffers and enzymes for qPCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Hiscript 2 q rt supermix for qpcr gdna wiper kit, by Vazyme (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Superrt cdna kit, by CWBIO (1 mentions)
Rnapure tissue kit, by CWBIO (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
4 protocols using ultrasybr mixture with rox kit
1
Quantitative RT-PCR for Ischemic Brain
2
Quantitative PCR Analysis of Drosophila Gene Expression
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The first-strand cDNA was synthesized with HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China) using an oligo(dT)18 primer and 500 ng total RNA template in a 10 μl reaction, following the instructions. Real-time qPCRs in the various samples used the UltraSYBR Mixture (with ROX) Kit (CWBIO, Beijing, China). The PCR was performed in 20 μl reaction including 4 μl of 10-fold diluted cDNA, 1μl of each primer (10 μM), 10 μl 2 × UltraSYBR Mixture, and 6 μl RNase-free water. The PCR conditions used were as follows: initial incubation at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 60°C for 45 s. N. lugens 18S rRNA or Drosophila rp49 were used as an internal control (S1 Table ). Relative quantification was performed via the comparative 2−△△CT method [104 (link)]. We used Drosophila heads to analyze the expression of Dsk and GAL4 in starved or refed condition.
3
Quantification of mRNA Expression by qRT-PCR
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QRT-PCR for mRNAs was performed as described previously.18 (link) Total RNA was extracted from PCa cells with Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed according to the manual of the PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan). Real-time quantitative PCR was tried out with Ultra SYBR Mixture (with ROX) kit (CWBIO, Beijing, China) on the LightCycler 480II (Roche Applied Science, Basel, Switzerland) instrument. The Ct values were normalized using β-actin or RNU6 as an internal control to estimate the different expression of genes. Relative mRNA expression was calculated using the 2−ΔΔCt method. Each sample was run in triplicate to ensure quantitative accuracy. All sequences of synthetic oligonucleotides are listed in Supplementary Table 2
4
cDNA Synthesis and Real-Time qPCR Protocol
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The first-strand cDNA was synthesized with HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China) using an oligo(dT)18 primer and 500 ng total RNA template in a 10 μl reaction, following the instructions.
Real-time qPCRs in the various samples used the UltraSYBR Mixture (with ROX) Kit (CWBIO, Beijing, China). The PCR was performed in 20 μl reaction including 4 μl of 10-fold diluted cDNA, 1μl of each primer (10 μM), 10 μl 2 × preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted December 15, 2020. ; https://doi.org/10.1101/2020.12.14.419390 doi: bioRxiv preprint UltraSYBR Mixture, and 6 μl RNase-free water. The PCR conditions used were as follows: initial incubation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 s and 60˚C for 45 s. N. lugens 18S rRNA or Drosophila rp49 were used as an internal control (Supplementary Table S1). Relative quantification was performed via the comparative 2 -△△CT method 98 .
Real-time qPCRs in the various samples used the UltraSYBR Mixture (with ROX) Kit (CWBIO, Beijing, China). The PCR was performed in 20 μl reaction including 4 μl of 10-fold diluted cDNA, 1μl of each primer (10 μM), 10 μl 2 × preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted December 15, 2020. ; https://doi.org/10.1101/2020.12.14.419390 doi: bioRxiv preprint UltraSYBR Mixture, and 6 μl RNase-free water. The PCR conditions used were as follows: initial incubation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 s and 60˚C for 45 s. N. lugens 18S rRNA or Drosophila rp49 were used as an internal control (Supplementary Table S1). Relative quantification was performed via the comparative 2 -△△CT method 98 .
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