We stored at ?80 °C. Complementary DNA (cDNA) synthesis

We studied assessed plasmasamples obtained at the time of initial diagnosis of 142 40 lymphoma DLBCL patients whowere diagnosed with DLBCL. All of these patients have undergone molecular andphenotypic classification with available clinical datainformation.  Moreover, 45 38 serum plasma samplesobtainedfrom healthy individuals. The blood sampleswere allowed to stand at room temperature for 30 min and then centrifuged at3,000 rpm for 15 min at 4°C. To remove remaining cellularcontaminants, serum plasma sampleswere subjected to additional centrifugation at 12,000 rpm for 10 min Bloodsamples of all patients and healthy control patients werecollected. The median follow-up was approximately 13.

78 months(range, 2-23 months). The study was approved by the Ethics Committee of ZahedanMedicalUniversity  of Medicalscience General Hospital, and ethicalpermission and informed consent were obtained from all contributors.  MicroRNAisolation and quantitative real-time PCR analysis (qRT-PCR)TotalRNA including miRNA and small RNA fraction was isolated using miRNeasySerum/Plasma Kit (Qiagen) according to the manufacturer’s protocol. The U6 snRNA was used as an internalcontrol.

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Quality and quantity of isolated RNA were evaluated by means ofNanoDrop 2000. Isolated RNA was stored at ?80 °C. Complementary DNA (cDNA) synthesis wascarried out utilizing miScript II RT Kit (Qiagen) according to themanufacturer’s protocol.

HiSpecbuffer (Qiagen) was used for the selective conversion of mature miRNAs. Afterreverse transcription, cDNA samples were stored at ?20 °C. For qT-PCR, themiScript® SYBR® Green PCR Kit (Qiagen) was used according to the manufacturer’sprotocol.Amplification curves plots wereanalyzed to define Ct (threshold cycle). The melting curve analysis wasimplemented.

The relative expression of plasma miRNA in DLBCL cases was analyzedwith the 2???Ctmethod, using pooled miRNA from healthy individualsas the reference24. Each sample was assessed in triplicate and the raw datawere indicated as the relative quantity of target miRNA and normalized with U6.RT-PCR primers: miR-155: F: 5′-GACTGTTAATGCTAATCGTGATAG-3′; R:  5′ GTGCAGGGTCCGAGGTATTC-3′, U6: F: 5′-GCGCGTCGTGAAGCGTTC-3′;R: 5′ GTGCAGGGTCCGAGGT-3.

 Statistical analysisAllstatistical analyses were carried out using the SPSS 20.0 software package(SPSS, Chicago, IL, USA). Pearson ?2 analysis or Fisher exact test was employedto compare the difference of categorical variables. miR-155 expression levels in serum samples were shown bymean and standard deviation (mean ± SD) and compared using student’s  t-test. The (Mann– Whitney independent t-test) two-tailed Chisquared test was employed toexplore the correlation between miR-21expression and clinical pathologicalfeatures. Survival curves for OS were estimated by using Kaplan-Meierand their 95% confidence intervals (CI) were calculated through log-Rankmethod.