Reference genes for quantitative RT-PCR data in gastric tissues and cell lines.
OBJECTIVE
To evaluate the suitability of reference genes in gastric tissue samples and cell lines.


METHODS
The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization.


RESULTS
ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44).


CONCLUSIONS
GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.
Fernanda Wisnieski, Danielle Queiroz Calcagno, Mariana Ferreira Leal, Leonardo Caires dos Santos, Carolina de Oliveira Gigek, Elizabeth Suchi Chen, Thaís Brilhante Pontes, Marília de Arruda Cardoso Smith, Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo 04023900, Brazil.
Fernanda
F
Wisnieski
Danielle Queiroz
DQ
Calcagno
Mariana Ferreira
MF
Leal
Leonardo Caires
LC
dos Santos
Carolina de Oliveira
Cde O
Gigek
Elizabeth Suchi
ES
Chen
Thaís Brilhante
TB
Pontes
Paulo Pimentel
PP
Assumpção
Mônica Barauna
MB
de Assumpção
Sâmia
S
Demachki
Rommel Rodríguez
RR
Burbano
Marília de Arruda Cardoso
Mde A
Smith
D000199:Actins / Q000235:genetics; D000230:Adenocarcinoma / Q000235:genetics*; D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D019295:Computational Biology; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D013274:Stomach Neoplasms / Q000235:genetics*; D001613:beta 2-Microglobulin / Q000235:genetics
D000199:Actins; D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human
Gastric cancer; Gene expression; Normalization; Quantitative real-time polymerase chain reaction; Reference gene