Computational protocol: Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

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Protocol publication

[…] Ten random colonies formed after plating CD44 knockdown BCSCs at low density were used for the analysis of gene expression. To evaluate the differentiated status of BCSCs, expression of 15 genes related to the properties of cancer stem cells and cancer/normal cells, as well as some genes related to signaling pathways over-expressed in cancer stem cells, were analyzed in comparison with BCSCs and non-BCSCs. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; [Genebank:NM_002046]) was used as an internal control for all experiments. All primers used in this study were designed using Primer Blast software (NCBI). Primer pairs were chosen to give polymerase chain reaction (PCR) products of 100-350 bp. The universal primer for each forward and reverse primer was then added. The universal primer sequence was suggested by the manufacturer, using the GenomeLab GeXP genetic analysis system (Beckman-Coulter, Brea, California). All primer sequences are listed in Table . All primers were checked for specificity and working status by in silico PCR and in vitro reverse-transcription PCR using a universal RNA template (Clontech, CA, USA). Only primer pairs that gave the intended PCR products were used in subsequent experiments. Two multiplex PCR reactions were used to evaluate the change in stemness: one multiplex with 17 genes included Bcl-2 [Genebank:NM_000633], Fos [Genebank: NM_005252], ICAM1 [Genebank:NM_000201], CCND1 [Genebank: NM_053056], MMP7 [Genebank:NM_002423], Myc [Genebank:NM_002467], PRKCE [Genebank:NM_005400], TP53 [Genebank:NM_000546], VCAM1 [Genebank:NM_001078], IL4R [Genebank:NM_000418], PTCH1 [Genebank:NM_000264], HSPB1 [Genebank: NM_001540], PTGS2 [Genebank: NM_000963], HSF1 [Genebank:NM_005526], LEF1 [Genebank:NM_016269], TCF7 [Genebank:NM_003202], and FASN [Genebank:NM_004104] and the other with five genes included Muc-1 [Genebank:NM_002456], cyclin E2 [Genebank:NM_004702], EGFR [Genebank:NM_005228], Myc [Genebank:NM_002467], and cyclin D1 [Genebank:BC001501].RNA was isolated from all cell samples using an RNA isolation kit (Fermentas, Glen Burnie, Maryland, USA). Gene expression levels of 15 genes involved in drug resistance, cell cycle and signaling pathways were assayed using the capillary GenomeLab GeXP genetic analysis system (Beckman-Coulter, Brea, California). A multiplex panel was designed to assess the genes. In addition to the genes of interest, each panel contained an internal control gene (kanamycin resistance, KanR) and a normalization gene (GAPDH). cDNA was synthesized from 500 ng total RNA using the GenomeLab GeXP Start Kit (Beckman-Coulter, Brea, California). PCR and multiplex detection were performed according to the manufacturer's instructions. GeXP data were analyzed after normalization of all genes of interest against the geometric mean of the normalization gene. […]

Pipeline specifications

Software tools Primer-BLAST, In-Silico PCR
Application qPCR
Organisms Mus musculus
Diseases Breast Neoplasms, Neoplasms, Brain Stem Neoplasms, X-Linked Combined Immunodeficiency Diseases
Chemicals Doxorubicin