The radiologist had been previously trained in using a transparent grid template to quantify the total breast area and the area composed of dense tissue.
Of the resulting cDNA, 30 ng/μl was used as the template to quantify the relative content of mRNA by real-time PCR (ABI PRISM 7700 sequence detection system, Applied Biosystems, Foster City, CA USA) using respective primers and SYBR Green.
A 1 10 dilution of the resulting cDNA was used as a template to quantify the relative content of mRNA by real-time PCR (ABI PRISM 7700 Sequence Detection System) using respective primers and SYBR Green.
For mRNA dosage studies, oligo-dT-primed cDNA was obtained using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then used as template to quantify CDK6 (Hs01026371_m1), MCL-1 (HSp1050896_m1) and Sp1 (Hs00412720_m1) levels by TaqMan assay (Applied Biosystems); normalization was performed with GAPDH (Hs03929097_g1).
The 1 μg of total RNA was transcribed into cDNA by using the iScripTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), and 40 ng/μl of the resulting cDNA was used as the template to quantify the relative content of mRNA by using QuantiTect SYBR Green PCR kit (Qiagen) with DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research, Waltham, MA, USA).
In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions.
The copy numbers of the standard template were determined to quantify EphB4 and ephrinB2 mRNA levels in samples for real-time reverse transcription PCR (RT PCR).
As a result, the Ct values can therefore be used as a measure of template DNA and to quantify the relative amount of 3895 bp deletion compared to wild-type mtDNA.
We performed a number of control experiments to quantify templates and assess the accuracy of estimates of frequencies of single nucleotide polymorphism (SNP).
To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation.
