Rotor gene q 5plex hrm real time pcr

Total RNA was isolated from the untransfected, transfected, and BTZ-treated RPMI 8226 and
U266 cells according to the TRIzol manufacturer’s protocol (InvitrogenTM, USA). A total of
2000 ng of RNA was reverse transcribed using specific miRNA stem-loop primers from Qiagen
for miR-19a to generate cDNA using a Hyperscript Reverse Transcriptase First-strand
Synthesis kit (GeneAll Biotechnology Co., Ltd., Korea). Snord47 was used as the internal
control for normalization of miRNA expression. Quantitative real-time polymerase chain
reaction (qRT-PCR) was performed with a SYBR® Premix Ex Taq™ miRNA RT-qPCR Detection Kit
(Takara, USA, cat. no. RR820Q) using a Qiagen Rotor-Gene Q 5PLEX HRM Real-Time PCR. Table
1 shows the primer sequences used in this experiment. The PCR program cycling parameters
were: 95˚C for 15 seconds, 58˚C for 30 seconds, and 72˚C for 30 seconds for 45 cycles.
Data analysis was done by 2^-ΔΔCT to calculate the fold change for relative
miR-19a expression compared to the untreated control.

Total RNA was isolated from the untransfected, transfected, and BTZ-treated RPMI 8226 and
U266 cells according to the TRIzol manufacturer’s protocol (InvitrogenTM, USA). A total of
2 μg of total RNA was reverse transcribed into cDNA using a Hyperscript Reverse
Transcriptase First-strand Synthesis kit with oligo-dT primers in accordance with the
manufacturer’s instructions (GeneAll Biotechnology Co., Ltd., Korea) for evaluation of the
SOCS3, STAT3, BCL-2, CDKN1A and PTEN genes.
β-Actin was used as the internal control. qRT-PCR was performed with
the SYBR® Premix Ex Taq™ miRNA RT-qPCR Detection Kit (Takara, USA, cat. no. RR820Q) using
a Qiagen Rotor-Gene Q 5PLEX HRM Real-Time PCR. The PCR program cycling parameters were:
95˚C for 15 seconds, 58˚C for 30 seconds, and 72˚C for 30 seconds for 45 cycles. Data
analysis was performed by 2^-ΔΔCT to calculate the fold changes for the relative
expressions of the above genes compared to the untreated control.