Liver tissue was flash frozen and used for RNA extraction using a standard TRIzol reagent protocol (Molecular Research Center, Cincinnati, OH). Quantitative, real-time PCR using Fluorescein amidites-labeled primers specific for CHOP and PERK (Applied Biosystems, Grand Island, NY) was performed using a Stratagene Mx 3000P and/or Mx 3005P (Agilent, Santa Clara, CA) and normalized to the reference gene Rpl18.
Trizol reagent protocol
Manufactured by Molecular Research Center
Sourced in United States
TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA from various biological samples. The reagent maintains the integrity of RNA during sample homogenization or lysis.
Automatically generated - may contain errors
3 protocols using trizol reagent protocol
1
Quantitative PCR Analysis of CHOP and PERK
2
Quantifying MUC1 Gene Expression in Mice
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The skin near the injection site was taken from immunized mice and lysed using a
homogenizer. Total RNAs were extracted from lysates in the presence of RNase inhibitors
according to the TRIzol reagent protocol (Molecular Research Center, OH). RNAs were
dissolved in diethyl pyrocarbonate (Sigma-Aldrich, MO)-treated water. cDNA was generated
from an mRNA template using a 15-mer poly-dT oligonucleotide (Invitrogen, CA) and
superscript reverse transcriptase enzyme (GIBCO-BRL, CA) at 37°C for 1 h using the
SuperScript Preamplification System protocol. MUC1 gene expression was
detected by PCR using the same primers and protocols as described above.
homogenizer. Total RNAs were extracted from lysates in the presence of RNase inhibitors
according to the TRIzol reagent protocol (Molecular Research Center, OH). RNAs were
dissolved in diethyl pyrocarbonate (Sigma-Aldrich, MO)-treated water. cDNA was generated
from an mRNA template using a 15-mer poly-dT oligonucleotide (Invitrogen, CA) and
superscript reverse transcriptase enzyme (GIBCO-BRL, CA) at 37°C for 1 h using the
SuperScript Preamplification System protocol. MUC1 gene expression was
detected by PCR using the same primers and protocols as described above.
3
Carbohydrate and Protein Analysis of Buds
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To conduct a physiological analysis and the growth quality assay, the buds were dried individually by freeze dryer (333Bi5, DorsaTech Co., Iran) at -80°C, for 24 h. For the total carbohydrate content, 100 mg of dried powder was extracted using ethanol 80% for 60 min. Five mL of HCl 1.1% was added into the supernatant, and the mixtures were heated in a water bath (97°C) for 30 min. The samples (1 mL) were mixed with 5 mL of ice-cold anthrone reagent (72% sulfuric acid containing 0.2% anthrone) and reheated (11 min). The solutions absorbance was measured at 630 nm. The total protein content of 0.1 g of the bud tissues were extracted over TRIzol reagent protocol (Molecular Research Center, Inc., Cincinnati, OH, USA). Thereafter, the protein concentration was determined according to the Bradford assay kit (BioRad Co., Hercules, CA, USA) in the comparison with BSA as the standard [21, 22] .
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