Computational protocol: Donor‐Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte‐Like Cells

Similar protocols

Protocol publication

[…] Primers for use in quantitative reverse transcription (qRT)‐PCR gene expression studies were designed using the NCBI Primer BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer quality was assessed using OligoCalc and purchased from Eurofins (Supporting Information Table S4). Samples were collected in QIAzol (QIAgen), and extracted using the miRNeasy extraction kit according to the manufacturer's protocol (QIAgen) Extracted RNA content was measured using the NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA was reverse transcribed to complementary DNA (cDNA) using the ImProm‐II reverse transcription kit (Promega).Due to low RNA yield following extraction, whole transcriptome amplification was performed on selected samples using the QuantiTect Whole Transcriptome Kit (Qiagen), following the manufacturer's protocol. Briefly, 100 ng of starting RNA from each sample was mixed with reverse transcriptase kit component and incubated at 37°C for 30 minutes and 95°C for 5 minutes. Ligation mix was then added to each sample and incubated for 2 hours at 22°C. Finally, the amplification mix was added and incubated for 8 hours at 30°C and 5 minutes at 95°C. Temperature controlled steps were carried out using the GeneAmp PCR system 9700 thermal cycler system (Applied Biosystems, Waltham, MA, www.thermofisher.com) and amplification confirmed using the NanoDrop spectrophotometer.Gene expression analysis was performed using the SYBRGreen JumpStart Taq ReadyMix (Sigma Aldrich). Briefly, 10–100 µg of cDNA was amplified by qRT‐PCR as follows: 95°C for 10 minutes, then 40 cycles for 95°C 15 seconds, and 60°C for 60 seconds using the ViiA7 qRT‐PCR machine (Applied Biosystems). Results were calculated using the CT values generated, normalized against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and succinate dehydrogenase and calculated relative to a calibrator sample (e.g., PHH) using 2−ΔΔCT method . Sendai virus expression analysis of iPSCs was conducted using the TaqMan iPSC Sendai Detection Kit (Life Technologies) according the manufacturer's instructions. Results were normalized using GAPDH and calculated relative to the reprogramming plate using 2−ΔΔCT method. […]

Pipeline specifications

Software tools Primer-BLAST, OligoCalc, ViiA
Application qPCR
Organisms Homo sapiens
Diseases Chemical and Drug Induced Liver Injury