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

We studied assessed plasma
samples obtained at the time of initial diagnosis of 142 40 lymphoma DLBCL patients who
were diagnosed with DLBCL. All of these patients have undergone molecular and
phenotypic classification with available clinical data
information.  Moreover, 45 38 serum plasma samples
obtained
from healthy individuals. The blood samples
were allowed to stand at room temperature for 30 min and then centrifuged at
3,000 rpm for 15 min at 4°C. To remove remaining cellular
contaminants, serum plasma samples
were subjected to additional centrifugation at 12,000 rpm for 10 min Blood
samples of all patients and healthy control patients were
collected. The median follow-up was approximately 13.78 months
(range, 2-23 months). The study was approved by the Ethics Committee of Zahedan
Medical
University  of Medical
science General Hospital, and ethical
permission and informed consent were obtained from all contributors.

 

MicroRNA
isolation and quantitative real-time PCR analysis (qRT-PCR)

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Total
RNA including miRNA and small RNA fraction was isolated using miRNeasy
Serum/Plasma Kit (Qiagen) according to the manufacturer’s protocol. The U6 snRNA was used as an internal
control. Quality and quantity of isolated RNA were evaluated by means of
NanoDrop 2000. Isolated RNA was stored at ?80 °C. Complementary DNA (cDNA) synthesis was
carried out utilizing miScript II RT Kit (Qiagen) according to the
manufacturer’s protocol. HiSpec
buffer (Qiagen) was used for the selective conversion of mature miRNAs. After
reverse transcription, cDNA samples were stored at ?20 °C. For qT-PCR, the
miScript® SYBR® Green PCR Kit (Qiagen) was used according to the manufacturer’s
protocol.

Amplification curves plots were
analyzed to define Ct (threshold cycle). The melting curve analysis was
implemented. The relative expression of plasma miRNA in DLBCL cases was analyzed
with the 2???Ctmethod, using pooled miRNA from healthy individuals
as the reference24. Each sample was assessed in triplicate and the raw data
were 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 analysis

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