Subjects: Specifically, in this formula, Ct represents


female handball players from the Helwan
University team constituted
subjects of this study. Written informed consent was obtained from all
participants according to the ethical approval for this study conformed to the
standards of the Declaration of Helsinki. 
The Helwan
University review board
approved the protocol before study initiation. Height, weight and age were
recorded for all investigated subjects. All participants were required to
abstain from non-steroidal anti-inflammatory use for at least 7 days prior the
experimental study day.

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The subjects trained for 8 weeks as 3 days/week in a handball
training program. In the first three weeks, training session were performed
once daily but raised to be twice daily in the second four weeks. Each training
session consisted of 10 min. warm up followed by 90 – 120 min. of specific handball
skills training program under specialized coach supervision, then positive cool
down for another 10 min.  Proposed
handball game was played at the days of sampling. Then tapering sessions were
applied to the players as decreasing in training intensity by about 20% and
sessions performed as once daily again for five days according to Expert
opinion Then, an official game was played for the second sampling collection.


Plasma sampling:

milliliters of blood was collected under aseptic conditions in standard
anticoagulant (EDTA)-treated vacutainer tubes at baseline (prior to exercise
testing) and immediately post-exercise testing. All blood samples were
centrifuged at 2000 g for 10 min to pellet cellular elements immediately
after each blood draw. The supernatant plasma was then separated and
immediately frozen at –80?C
till PCR assay.



miRNA extraction:

            Total RNA
extraction was performed using a MicroRNA Extraction Kit (Shanghai ShineGene
Molecular Biotech, Inc., Shanghai,
Quantitative efficacy and reproducibility of c-miRNA extraction was confirmed
by extraction via serial dilutions.


Quantification of microRNA expression:

To quantify levels of select c-miRNA, standard reverse
transcription-quantitative (real time) polymerase chain reaction (RT-PCR) was
utilized. Specifically, reverse transcription was performed to generate cDNA
representing levels of mature c-miRNA molecules.


Fold-change of both miRNA 1 and miRNA 206 species were
calculated using the formula specified by the manufacturing company.
Specifically, in this formula, Ct represents the ‘real time’ cycle
number at which the increase in miRNA probe fluorescence is exponential. As
reference control for miRNA quantification, Ct values were then
subtracted from Ct values obtained from exogenously added miR-1 or
miR-206, as described above. Ct values were then compared (?Ct) with
each athlete’s own resting baseline at the pre-tapering time point (normalized
to fold-change of 1).

Handball skills Measurements:

Handball skills were assessed before and after the program and after
tapering period as follow:


Statistical analysis:

A Computer
program (SPSS version 17) was used
to analyze obtained results. Student’s Mean,
standard deviation of players and ANOVA test were used to examine the difference between baseline, after program
and after tapering measurements before and after the training sessions. Probability
was assigned significant at ? 0.05.

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