Prior to cDNA preparation, all samples were stored in RNAlater (Qiagen, Hilden, Germany) at −20°C. Where tissues were processed, samples were disrupted using a TissueLyser LT (Qiagen). RNA was extracted from samples using RNeasy spin columns (Qiagen) and extracted nucleic acids were subjected to DNaseI (Qiagen) treatment in solution and a further column cleanup. RNA for qRT-PCR was reverse transcribed using the Applied Biosystems (Carlsbad, CA, USA) high capacity reverse transcription kit with an added RNase-inhibitor (Promega Biosciences, Madison, WI, USA) and cDNA was cleaned using QIAquick spin columns (Qiagen). All elutions were conducted with nuclease-free water (Qiagen).
Purified cDNA was used as template for the amplification of target gene transcripts with SYBR Green PCR master mix (Applied Biosystems) using the ABI Prism SDS 7000 and 7900HT machines (Applied Biosystems). Target gene expression was determined relative to Hprt using the ΔCT method using previously-described primer sets and methodology [21 (link)]. In plots showing expression, a hashed line indicating the theoretical detection limit is shown. Fold change values are calculated against an unstimulated control, represented by the hashed line, which is standardized to 1.
Purified cDNA was used as template for the amplification of target gene transcripts with SYBR Green PCR master mix (Applied Biosystems) using the ABI Prism SDS 7000 and 7900HT machines (Applied Biosystems). Target gene expression was determined relative to Hprt using the ΔCT method using previously-described primer sets and methodology [21 (link)]. In plots showing expression, a hashed line indicating the theoretical detection limit is shown. Fold change values are calculated against an unstimulated control, represented by the hashed line, which is standardized to 1.
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