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Bioscience Methods 2014, Vol.6, No.1, 1-13
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12
Reverse transcription reactions with template-free
were used as a negative control to exclude DNA
contamination. Using a standardised procedure, all
PCRs were optimised with respect to the denaturing
temperature and extension time to yield a product of
the expected size.
3.5 Real-time quantitative reverse transcription-PCR
(qRT-PCR) for
StOSM
expression analysis
Total RNA was treated with RNase-free DNase
(Qiagen). The quantity and quality of the RNA
samples were estimated using a Nano
spectrophotometer (ND-1000; Thermo Scientific).
RNA samples with a 260:280 ratio ranging from 1.9 to
2.1 and a 260:230 ratio ranging from 2.0 to 2.5 were
used for the qRT-PCR analyses.
Real-time quantitative reverse transcription-PCR
analysis was performed using the PrimeScript™ RT
Master Mix and SYBR® Premix Ex Taq™ II
(RR820A and RR036A, TaKaRa Biotechnology,
Dalian, China) following the manufacturer’s protocol.
Briefly, 50-ng aliquots of the total RNA template were
subjected to each qRT-PCR in a final volume of 20 µL
containing 4 µL of the PrimeScript RT Master Mix
and 0.4 µL of qScript Reverse Transcriptase along
with target-specific primers (200 nm).
All reactions were performed in triplicate using 1 μl of
template cDNA in a final volume of 20 μl in a
Bio-Rad Real Time PCR System (Bio-Rad, CA, USA).
The Illumina Eco qPCR machine with fast
quantitative PCR cycling parameters was used for
complementary DNA synthesis with the following
conditions: 37°C for 15 min followed by 85°C for 5 s;
initial denaturation − 95°C for 30 s for 1 cycle; 2 step
PCR − 95°C for 5 s and 60°C for 30 s for 40 cycles.
Beta-actin (accession number DQ252512) was used as
an internal control. The relative gene quantification
method was used to calculate the expression levels of
different target genes (Livak and Schmittgen 2001).
Primer specificity was determined by melting curve
analysis and qualification analysis by RT-PCR. A
standard curve was generated based on serial dilutions
of cDNA to calculate the gene-specific PCR.
The relative expression levels of each
StOSM
gene
were analysed using the IQ5 software and are
presented as the fold-difference from the baseline
expression levels. Using Origin Pro 8.0 software
(Origin Lab Corp., Northampton, MA, USA)
，
paired
t-tests were performed to assess the significance of
expression level differences between the treatments
and the controls.
Author's contributions
YXL designed the experiment and primers used, performed
data analysis, wrote and revised the manuscript and supervised
performance of the project.
Performed the experiments: Qiao
LB, Dai HX, Fan HH, Zhang XP and Wu F. Analysed the data:
Yao XL, Qiao LB, and Fan HH. Wrote the paper: YAO XL and
Qiao LB.
Acknowledgments
This work was jointly supported by National Natural Science
Foundation of China (NSFC
31360275
, 31160236, To Yao XL).
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