RNA was isolated from all the samples by using GeneJET RNA purification kit (Thermo Scientific, Lithuania, EU) as per manufacturer’s protocol. RNA quantity was measured by absorbance ratio at 260/280 nm and integrity was verified on 1% agarose gel. The mRNA expression of EMT markers such as Vimentin (VIM), Fibronectin (FN) and E-cadherin (E-CAD) and matrix metalloprotease 9 (MMP9) was analyzed by qRT-PCR using iTaq Universal SYBR Green One-Step RT-PCR kit (BioRad Laboratories Inc., USA) and 7500 Fast Real-Time PCR System (Applied Biosystems, California, USA) using β-Actin as control house-keeping gene. The mRNA expression was quantified using the 2−ΔΔCt and presented as the mean ± SEM as fold change compared to controls (150 or 600μm microtumors). Primer sequences are given in Supplementary Table S1 .
Itaq universal sybr green one step rt pcr kit
Manufactured by Bio-Rad
Sourced in United States
The ITaq Universal SYBR Green One-Step RT-PCR kit is a reagent kit designed for reverse transcription and real-time PCR amplification of RNA targets. It contains a ready-to-use master mix formulation, including all necessary components for reverse transcription and PCR in a single tube.
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2 protocols using itaq universal sybr green one step rt pcr kit
1
Quantifying EMT Markers in Microtumors
2
Quantification of Liver mRNA Expression
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Total RNA was isolated from PND35 livers using the Thermo Fisher PureLink RNA Mini Kit according to the manufacturer’s instructions and quantified with a NanoDrop spectrophotometer. Primers reported from the literature were checked by NCBI Primer Blast for accuracy. Primers were purchased from Integrative DNA Technologies as RxnReady Oligos and diluted to a 1 mM stock. Expression of mRNA was quantified by qRT-PCR using the BioRad iTaq Universal SYBR Green One-Step RT-PCR Kit containing 300 nM primer and 50 ng/µL of total RNA in a 10 µL reaction. PCR reaction products were verified by melting temperature or gel electrophoresis and compared to expected band size. mRNA expression relative to Gapdh or Actb was normalized to WT levels.
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