Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.
In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.
Mogal
Ashish
A
Department of Pathology, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA.
Abdulkadir
Sarki A
SA
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008297:Male; D051379:Mice; D011467:Prostate / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D012337:RNA, Ribosomal, 18S; C402487:RPL13A protein, human; D012269:Ribosomal Proteins; C520792:Rpl13a protein, mouse; C012589:trichostatin A; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases