Sybr green jumpstart taq readymix for qpcr

mRNA abundance was determined using cDNA prepared from RNA, isolated from duodenal biopsies. Real-time PCR assays were performed using SYBR Green JumpStart Taq ReadyMix for QPCR (Sigma Aldrich). Assays were performed in triplicate using a Rotorgene 3000 (Qiagen, Crawley, UK) and relative abundance was calculated using RG-3000 comparative quantification software. Real-time amplification of RNA polymerase IIA (POLR2A) was carried out simultaneously as the control reference (see Table S2 for qPCR primer sequences).

A pool of six placental samples obtained from at least two litters from the same group was used total RNA extraction. RNA was extracted with Trizol (Invitrogen Life Technology) according to the manufacturer’s instructions and treated with DNase I to eliminate genomic DNA contamination. RT was accomplished using a commercially available first-strand cDNA synthesis kit (QIAGEN). The RT reactions were performed following the manufacturer’s protocol to reverse transcribe 1 μg of total RNA. Real-time PCR was performed with the Bio-Rad CFX96 real-time PCR instrument (Bio-Rad, Hercules, CA) and SYBR Green JumpStart Taq ReadyMix for Q-PCR according to the manufacturer’s protocol (Sigma-Aldrich). Gene expression was normalized using the housekeeping gene Gapdh as the reference gene. The primer sequences for the genes analyzed are listed in Table S1, Table S2 and Table S3. Primer sequences were obtained from Primer Bank or designed using Primer Express. Samples were analyzed using the ΔΔCt method.

Total RNA was isolated from the cerebellum halves of 1-month-old mice using TRIzol® Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). After removing genomic DNA by DNase I treatment (Thermo Fisher Scientific Inc., Waltham, MA, USA), RNA concentration and purity were determined using NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). Isolated RNA (1 μg) was reverse transcribed into cDNA (RevertAid H Minus First Strand cDNA Synthesis Kit, Thermo Fisher Scientific Inc., Waltham, MA, USA). The obtained cDNA samples were diluted 20×. Each reaction for qPCR analysis contained 4 μl diluted cDNA, 5 μl SYBR Green JumpStart Taq ReadyMix for qPCR (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μl ultrapure water, 0.25 μl 10 mM forward primer, and 0.25 μl 10 mM reverse primer. The primer sequences are listed in Table S1. qPCR was performed with the initial activation at 94 °C for 120 s, followed by 39 cycles at 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s using the CFX384™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The –ΔΔCq method was used to quantify the relative mRNA expression [33 (link)] with Hprt1 as a reference gene [34 (link)]. The Isl1 reaction products were analyzed using agarose gel electrophoresis. Equivalent aliquots of each amplification reaction were separated on a 2 % agarose gel containing 0.5 μg/ml ethidium bromide.