High capacity cdna reverse transcription kits with rnase inhibitor

Twenty-one snap-frozen cholangiocarcinoma (CCA) specimens, including seven samples with tumor-adjacent normal tissues, were collected from the University of Alabama at Birmingham (UAB) Tissue Biorepository, after obtaining approval from the Institutional Review Board. All samples were stored in the Invitrogen RNAlater-ICE stabilization solution (Thermo Fisher Scientific, Waltham, MA, United States) until processed for RNA extraction and real-time quantitative PCR (qPCR).
Total RNA was isolated from CCA and normal specimens with TRIzol reagent using the manufacturer’s instructions (Invitrogen, Carlsbad, CA, United States). Total RNA (2 μg) was reverse transcribed using high-capacity cDNA reverse transcription kits with RNase inhibitor (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, United States). The cDNA (10-ng) samples were used for validation and quantification of four genes (MDK, HNF1B, PACS1, and GLUD1) using PowerUp SYBR green master mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, United States) on an ABI real-time PCR machine and analyzed with Quant-studio real-time PCR software (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, United States). The Student’s t-test with two-tailed distribution was used to calculate the p-values for the statistical significance.

The pKERs (p = 0; n = 5 animals) and pDFs (p = 1; n = 4–5 animals) were plated in 6-well plates at a density of 2.0 × 10⁶ or 1.0 × 10⁶ cells per well, respectively, in adequate culture media. Subconfluent (60–70% confluency) cultures were washed with PBS and incubated with CM-Hyp or CM-Nor diluted 1:1 in appropriate culture media (see Table 1) for 24 and 48 h. The ADSC-BM medium mixed with respective KERs’ and DFs’ media supplemented with 1% penicillin/streptomycin was used as a control. Total RNA was extracted using TRIzol^® Reagent (Invitrogen by Thermo Fisher Scientific Baltics UAB). Genomic DNA was removed from RNA samples using DNase I Amplification Grade kit (Sigma-Aldrich Co., St. Louis, MO, USA). Synthesis of complementary DNA with 1000 ng of RNA was performed using High-Capacity cDNA Reverse Transcription Kits with RNase Inhibitor (Applied Biosystems by Thermo Fisher Scientific Baltics UAB) according to the manufacturer’s specifications. Expression of specific mRNA was quantified with TaqMan Gene Expression Assays (Applied Biosystems by Thermo Fisher Scientific Baltics UAB) on an ABI ViiA™ 7-sequence detection system (Applied Biosystems by Life Technologies, Singapore). Gene names and primer-probe set information are presented in Supplementary Table S1. All results were normalized to HPRT1 expression as endogenous control using the PCR Miner algorithm [56 (link)].