Time Quantitative RT-PCR Research Articles

RNA-Seq Analysis Reveals Genetic Bases of the Flowering Process in Oriental Hybrid Lily cv. Sorbonne

Flowering is a critical step in plant reproduction. Oriental lily (Lilium) characterized by showy flowers is increasingly used; however, the blossom period is an important limiting factor influencing the value of lily. Improving the flowering of lily by molecular breeding technology has limitless applications, but the mechanism of the regulation of lily flowering remains to be studied. Transcriptomes providing comprehensive sequence profiling data of transcription variation during flowering process in Oriental hybrids lily Sorbonne were assembled from RNA Seq data. Approximately 124.16 million 90 bp paired end clean reads were assembled into 66 327 unigenes and compared with the UniProt databases. There are 30 254 unigenes that have significant hits to the sequences in the UniProt database, 60 738, 16 601, and 12 494 unigenes have sim ilarity to the GO, KEGG, and COG databases, respectively. By analyzing dynamic changes in the transcrip tome of lily flowering based on our RNA sequencing (RNA Seq) data, some genes involved in floral induc tion were found, which revealed the complicated flower regulation network at the transcriptome level during lily flowering. Moreover, 12 DEGs related to flowering including LoLFY, LoMAF, LoFT, LoAG, LoCBF, LoAGL6a, LoSOC1, LoSEP1, LoNAC1, LoAPX, LoARF10, and LoICE were identified with real time quan titative RT PCR analysis. The results suggested that the flower of Oriental lily possessed a high proportion of flowering genes active at different stages of flowering. According to the results of the present study, we pre dicted that they would play an important role during flowering process; these data provided the foundation for future studies of metabolism during flowering of Oriental lily.

WT1 Expression for Monitoring Minimal Residual Disease In Acute Lymphoblastic Leukemia Patients

AbstractAbstract 3234 Background.Wilms' tumor 1 (WT1) gene encodes a transcriptional factor involved in normal cellular development. It is over-expressed in most cases of acute myeloid leukemia (AML). Aim.To estimate the usefulness of WT1 expression quantitative assessment for monitoring MRD and for relapse prediction in acute lymphoblastic leukemia (ALL) patients. Methods.Real time quantitative RT-PCR was made according to the Europe Against Cancer Protocol. WT1 transcript level was determined in bone marrow (BM) samples of adult patients with AML (except acute promyelocytic leukemia) and ALL. We examined the samples of 77 patients with AML and 34 patients with ALL at diagnosis. We also analyzed 190 specimens of AML patients and 150 specimens of ALL patients during follow-up. The obtained WT1 copy numbers were normalized with respect to the β-GUS transcripts. Results.The median value of WT1 level at diagnosis was 581 (range 1,0-8462) in AML patients and 1161 (range 1,5-14879) in ALL patients (no statistical difference in Student's t-test). The median value of WT1 level of patients in complete remission was 4,3 (range 1,1-9,7) for AML and 5,2 (range 1,0-16,8) for ALL. At diagnosis 84% of AML patients and 61% of ALL patients showed the expression of WT1 one log higher than the median values in complete remission. In 7 assessed T-ALL patients the over-expression of WT1 at diagnosis was observed (the median value 1188, range 206–6972). The B-ALL patients with high WT1 expression at diagnosis (range 441–14879) and the B-ALL patients with low WT1 expression at diagnosis (range 1,5-63) differ in fusion genes. There were 2 patients with BCR-ABL (one p190 (7%) and one p210) within the B-ALL subgroup with high WT1 expression at diagnosis whereas within the B-ALL patients with low WT1 expression at diagnosis were 7 ones (6 patients with p190 (46%) and 1 with p210) (7% versus 46%, p=0,033 by Fisher Exact test). In the patients bearing a fusion gene transcript we performed a simultaneous analysis of WT1 and fusion gene transcript expression: in 19 patients with AML (8 AML1-ETO, 2 CBFβ-MYH11, 2 BCR-ABL, 1 DEK-CAN, 1 MLL-AF6, 1 MLL-AF9, 4 MLL duplications) and in 15 patients with ALL (9 BCR-ABL, 3 MLL-AF4, 1 MLL-ENL, 2 E2A-PBX1). It was found the parallelism between the dynamics of WT1 level and fusion-gene transcript level during follow-up. The haematological relapse was correlated with the increase of WT1 expression: 8 AML patients had a median of 1545 (range 140–2578) and for 6 ALL patients a median value was 1050 (range 31–2665) in relapse (no statistical difference in Mann-Whitney U test). We observed the increase of WT1 level was occurred earlier than the relapse was stated for AML patients and for ALL patients with high WT1 expression at diagnosis. Conclusion.The level of WT1 expression may be used as additional nonspecific marker for MRD detection and for imminent relapse prediction during follow-up in patients without additional molecular markers in ALL with high WT1 expression at diagnosis. Disclosures:No relevant conflicts of interest to declare.

Expression level of wild-type survivin in gastric cancer is an independent predictor of survival.

Survivin is a novel antiapoptotic gene in which three splicing variants have been recently cloned and characterized. Survivin has been found to be abundantly expressed in a wide variety of human malignancies, whereas it is undetectable in normal adult tissues. We aimed to study the expression of three survivin splicing variants in gastric cancer, and to evaluate the prognostic significance of the expression of survivin variants in gastric cancer. Real time quantitative RT-PCR was performed to analyze the expression of survivin variants in 79 paired tumors and normal gastric mucosa samples at the mRNA level. Proliferative and apoptotic activity was measured using Ki-67 immunohistochemical analysis and the TUNEL method, respectively. All the cases tested expressed wild-type survivin mRNA, which was not only the dominant transcript, but also a poor prognostic biomarker (P = 0.003). Non-antiapoptostic survivin-2B mRNA was correlated with tumor stage (P = 0.001), histological type (P = 0.004), and depth of tumor invasion (P = 0.041), while survivin-DeltaEx3 mRNA showed a significant association with apoptosis (P = 0.02). Wild-type survivin mRNA expression levels are of important prognostic value and significant participation of survivin-2B and survivin-DeltaEx3 is suggested in gastric cancer development.