RT-PCR data analysis NAGLUand GALNS in MPS IIIB and IVA skin fibroblast
Description
Total RNA was isolated from skin fibroblasts obtained from healthy (GM00613) and mucopolysaccharidosis IIIB (GM02931 and GM01426) and IVA (GM01259 and GM00958) donors. qPCR assay was performed to quantitate gene expression of NAGLU and GALNS genes using SYBR Green. File contains metadata, raw data, normalized data and fold change.
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Steps to reproduce
Cells: Human fibroblasts (wild type: GM00613; MPS IIIB: GM02931/GM01426: MPS IVA: GM01259/GM00958) were obtained from the Coriell Institute for Medical Research. Real-time PCR: Total RNA was isolated using the Monarch® Total RNA Miniprep Kit (New England Biolabs; USA). 0.5 µg of total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem; USA). For each sample, five reactions were run. Each reaction contained 100 ng of cDNA, forward and reverse primers (targeting NAGLU, GALNS and ß-Actin mRNA) at 0.1 µM final concentration, 10 µL of 2X master mix, and 0.4 µl ROX reference dye (DyNAmo HS SYBR Green qPCR Kit, Thermo Scientific #F-410L). qPCR reactions were performed in a QuantStudio3 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc) using the recommended protocol. Relative gene expression was calculated using the 2–ΔΔCq method.