Quantification of mRNA and protein levels was performed by RT-qPCR and western blotting respectively, as described previously [24 (link)]. Total RNA was extracted using Ambion Pure-Link kits (Thermo Fisher) and was reverse transcribed using QuantiNova RT kit (Qiagen) and random primers. Quantitative PCR was performed using Roche SYBR Green using a Qiagen Roto-Gene Q PCR machine (20’@95 °C;20’@62 °C;20’@72 °C). Primers sequences are described in Supplement Table 1. Data were normalised to total amount of RNA. Western blots were performed using a Mini-Protean II system. Proteins were transferred to PVDF membrane using a semi-dry Turbo blotter system (Bio-Rad) and detected using ECL and a digital ChemiDoc imaging system (Bio-Rad). Phos-tag gels were prepared containing 100 μM Phos-tag acrylamide and 20 μM MnCl2 according to the manufacturer's instructions (Alpha Laboratories).
Quantinova rt kit
Manufactured by Qiagen
The QuantiNova RT kit is a reverse transcription kit designed for the conversion of RNA to cDNA. It includes all necessary components for efficient and sensitive reverse transcription reactions.
Automatically generated - may contain errors
Lab products found in correlation
Roto gene q pcr machine, by Qiagen (1 mentions)
Ambion pure link kit, by Thermo Fisher Scientific (1 mentions)
Semi dry turbo blotter system, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Sybr premix dimmer eraser kit, by Takara Bio (1 mentions)
Beyozol, by Beyotime (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Omniscript rt kit, by Qiagen (1 mentions)
Topreal qpcr 2x premix, by Enzynomics (1 mentions)
4 protocols using quantinova rt kit
1
Quantification of mRNA and Protein Levels
2
Quantitative Analysis of TRPC1 Expression
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Total RNA was isolated from A549, A549/CDDP, H460, and H460/CDDP cells using Beyozol (Beyotime Institute of Biotechnology) and subsequently reverse transcribed into cDNA using the QuantiNova RT Kit according to the manufacturer's protocol (Qiagen GmbH). qPCR was performed using the SYBR® Premix DimmerEraser™ kit (Takara Bio, Inc.). The thermocycling conditions were: 95°C for 30 sec; followed by 40 cycles of 95°C for 5 sec and 61°C for 30 sec. The expression of TRPC1 was assessed using the 2−ΔΔCq method and GAPDH was used as the internal control (17 (link)). The sequences of the primers were: TRPC1 forward, 5′-ACCTTCCATTCGTTCATTGG-3′ and reverse, 5′-TGGTGAGGGAATGATGTTGA-3′; and GAPDH forward, 5′-GAGTCCACTGGCGTCTTCAC-3′ and reverse, 5′-ATCTTGAGGCTGTTGTCATACTTCT-3′.
3
RNA Extraction and Real-Time PCR Analysis
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RNA was prepared from snap-frozen tissue using RNeasy mini kit (Qiagen). QuantiNova RT kit (Qiagen) and Omniscript RT kit (Qiagen) was used to synthesize cDNA from 1 μg of RNA. For RT-PCR reactions, cDNA samples were amplified with Hotstar Taq DNA polymerase (Qiagen). Quantitative RT-PCRs were carried out with Applied Biosystems real-time PCR 7500 using iTaq Universal SYBR green supermix (Bio-Rad Lab). Data were represented as the relative expression of gene after normalizing to β-actin. All primer pairs have been previously described (Bartholin et al., 2008 (link); Basciani et al., 2004 (link); Bopp et al., 2013 ; Puche et al., 2013 (link); Stewart et al., 2014 (link); Xiang et al., 2012 ; Yang et al., 2013 (link)).
4
qRT-PCR Analysis of Testicular Sly-KD Mice
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Testicular samples from Sly-KD mice were prepared as previously described (33 (link)). Total testis RNA was reverse transcribed using a QuantiNova RT kit (Qiagen) according to the manufacturer’s instructions. qRT-PCR was carried out using TOPreal qPCR 2X premix (Enzynomics) with primers listed in table S6. Amplifications were performed in at least triplicate for each sample. Relative gene expression levels were evaluated using the 2−ΔΔCt method (50 (link)) and were normalized to the level of Gapdh mRNA. Data are presented as means ± SD. Statistical analyses were performed using two-tailed Student’s t tests, and P ≤0.05 was taken as indicating a significant difference.
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