Faststart universal sybr green master mix rox

Snap-frozen half brains were homogenized using Lysing Matrix D tubes (MP Biomedicals, Cambridge, United Kingdom) and a Ribolyser tissue homogenizer (Bio-Rad Laboratories, Watford, United Kingdom). Total RNA was extracted using RNABee (AmsBio, Abingdon, United Kingdom), purified using an RNeasy Mini kit (Qiagen, Manchester, United Kingdom), and treated with RNase-free DNase I (Promega, Southampton, United Kingdom) to remove genomic DNA. First-strand cDNA synthesis was performed using 1 μg total RNA and the SuperScript III Reverse Transcriptase (Life Technologies, Waltham, MA, United States), and mRNA amplified using Oligo DT (Promega). RT-qPCR was then performed using the primers listed in Table 1 and FastStart Universal SYBR Green Master mix (Rox; Sigma-Aldrich) on an MX3005P RT-qPCR system (Agilent Technologies LDA UK Ltd., Stockport, Cheshire, United Kingdom). Cycle threshold values were analyzed using MxPro software (Agilent Technologies LDA UK Ltd.) and normalized relative to the reference gene Rpl19 using the ΔΔCT method. Expression values were normalized so that the mean level in the 1xPBS-treated controls was 1.0.

Total RNA was extracted from cell lysates with the RNeasy® Mini Kit (Qiagen, cat no.: 74104) following the manufacturer’s instructions. DNase I treated total RNA samples (1 μg) were subject to first-strand DNA synthesis by High Capacity cDNA Synthesis kit (ThermoFisher Scientific, cat no.: 4368814). Quantitative RT-qPCR was performed using FastStart Universal SYBR Green Master (Rox) mix (Sigma-Aldrich, cat no.: 4913850001) following manufacturer’s protocol. The reactions were performed in duplicates and run on the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The expression levels of genes were measured using the primers listed below. Primer design was done with Primer3 online tool [67 (link)]. The results were normalized against GAPDH. The analysis was performed in StepOne Software v2.2.2 and GraphPad prism and differential expression was calculated using unpaired t-test with Welch’s correction (p < 0.05 was considered significant).
The expression levels of the genes were measured using the following primers:
GAPDH F: 5′-GTCAGCTGTTGTTGGACCTG-3′,
GAPDH R: 5′-GGTCACCCCATCGAAGATAC-3′,
ZNF700 F: 5′-CACCCAGGAAGAGTGGACAT-3′,
ZNF700 R: 5′-ATGCCTTGTGTCCAGTGTCA-3′
HOXA3 F: 5′-′TGCCCTTCTGATCCTTTTTG-3′,
HOXA3 R: 5′-AATGCCAGCAACAACCCTAC-3′,
HOXB4 F: 5′-CTGGATGCGCAAAGTTCAC-3′,
HOXB4 R: 5′-AGCGGTTGTAGTGAAATTCCTT-3′,