To extract RNA, 50 mg tumor tissue was mechanically homogenized in 1 ml Qiazol (Qiagen, Hilden, Germany), whereas cells were lysed in 1 ml Qiazol by resuspending. 200 µl of chloroform were added, samples were mixed and incubated for 3 min followed by centrifugation for 15 min at 4 °C. The upper aqueous phase was added to 500 µl isopropanol to precipitate RNA. The pellet was washed with 75% ethanol, dried and solved in RNase free water. For reverse transcription 2 µg RNA were translated into cDNA using the “RNA to cDNA EcoDry” Kit (Takara Bio USA, Kusatsu, Japan) according to manufacturer’s instructions. The following qPCR analyses were performed in triplicates using the “Precision FAST MasterMix with ROX” (Primer Design, Southampton, UK), the respective Quantitect Primer pairs for detection of specific mRNAs (Qiagen, Hilden, Germany), and StepOnePlusTM qPCR system (2−ΔΔCT method). Values were normalized to endogenous housekeeping genes (RPL7 or RPLP0).
Precision fast mastermix with rox
Manufactured by Primerdesign
Sourced in United Kingdom
Precision FAST MasterMix with ROX is a ready-to-use, 2X concentrated master mix designed for fast, real-time PCR. It contains all the necessary components for FAST real-time PCR, including a hot-start DNA polymerase, dNTPs, MgCl2, and stabilizers. The master mix also includes ROX passive reference dye for normalization of the fluorescent signal.
Automatically generated - may contain errors
Lab products found in correlation
Rna to cdna ecodry kit, by Takara Bio (1 mentions)
Qiazol, by Qiagen (1 mentions)
Ecodrytm premix kit, by Takara Bio (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Mirneasy tissue cells advanced mini kit, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Nanophotometer np80, by Implen (1 mentions)
2 protocols using precision fast mastermix with rox
1
RNA Extraction and qPCR Analysis
2
Comprehensive miRNA and mRNA Analysis
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Both miRNA and total RNA were extracted by miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen) following the manufacturer's instruction. MicroRNA and RNA quantification was performed using a NanoPhotometer NP80 (Implen, Germany). MicroRNAs were reverse-transcribed by miScript Ⅱ RT kit (Qiagen), and cDNA was obtained using the RNA to cDNA Ecodry TM Premix Kit (Takara Bio Europe, France) according to the manufacturer's protocol. Quantitative polymerase chain reaction (qPCR) analyses were performed in triplicates using the Precision FAST MasterMix with ROX (Primer Design, UK). QuantiTect primers for detecting RNA encoding mouse MMP-2, MMP-9, MMP-14, and ADAM12 were obtained from Qiagen. We used XS13 as a housekeeping gene with primers (fwd-TGGGCAAGAACACCATGATG, rev-AGTTTCTC-CAGCTGGGTTG) purchased from Microsynth (Balgach, Switzerland). MiScript SYBR Green PCR kit (Qiagen) was used to analyze the miRNA expression, and RNU6 (Qiagen) was used as a housekeeping gene. A StepOnePlus TM qPCR system (Thermo Fisher Scientific, USA) was employed for the RT-PCR. The relative gene expression was calculated by 2 -ΔΔCT methods.
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