Sybr advantage qpcr premix

Reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) analysis was performed as described previously^{64 (link)}. Reactions were performed in triplo for determination of the levels of Yes, Lyn, Frk, Lck, Fgr, Hck, Blk and CyclofilinA (CF; control) in A431 cells. Cells were grown in complete medium and lysed in RNA-Bee (Tel-test Inc.) or Trizol reagent (Ambion #15596018), following manufacturer’s instructions. Total RNA was separated from DNA and proteins by the addition of chloroform (Sigma-Aldrich) and subsequent centrifugation at 12,000 × g for 15 min at 4 °C. The RNA was precipitated with isopropanol and washed with 70% ethanol. Integrity of the isolated RNA was assessed by agarose gel electrophoresis.
First strand cDNA synthesis was performed with 3 μg of total RNA using the first strand cDNA synthesis kit K1612 (Thermo Fisher Scientific) following manufacturer’s directions. The PCR reactions were run using SYBR Advantage qPCR premix (Clontech) on a 7500 Fast Real-Time PCR system (Applied Biosystems) and the primers (IDT) which were used, are shown in Table S2. Relative mRNA quantities were obtained using the 2^−ΔCt method. The ΔCt is the obtained Ct value of the SFK minus the obtained Ct value of CF (endogenous control).

RNA was treated with RNase-free DNase I (Qiagen) and reverse-transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Real-time quantitative reverse-transcription PCR (qRT-PCR) was performed to analyze mRNAs using an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and expression was normalized to that of RNU6B (U6) with SYBR green I (SYBR Advantage qPCR Premix; Clontech Laboratories, Mountain View, CA, USA). The following PCR primers were used: E-cadherin forward: 5′-TGCACCAACCCTCATGAGTG-3′ and reverse: 5′-GTCAGTATCAGCCGCTTTCAG-3′; Snail forward: 5′-TTCTCACTGCCATGGAATTCC-3′ and reverse: 5′-GCAGAGGACACAGAACCAGAAA-3′; N-cadherin forward: 5′-TCGCCATCCAGACCGACCCA-3′ and reverse: 5′-TGAGGCGGGTGCTGAATTCCC-3′; vimentin forward: 5′-CCTGAACCTGAGGGAAACTAA-3′ and reverse: 5′-GCAGAAAGGCACTTGAAAGC-3′; U6 forward: 5′-CTCGCTTCGGCAGCACA-3′ and reverse: 5′-AACGCTTCACGAATTTGCGT-3′. Expression of mature miRNA-622 was assessed using the TaqMan Human MicroRNA Assay Kit (Applied Biosystems) and normalized to the expression of U6.

For RNA extraction, Trizol (Life Technologies, Cat. # 15596018) was used according to the manufacturer’s protocol. About 200 ng of RNA was reverse-transcribed using Superscript II (Life Technologies, Cat. # 18964–014). StepOne Real Time PCR System (Life Technologies) and SYBR Advantage qPCR Premix (Clontech, Cat. # 638320) was used for quantification of cDNA. The oligonucleotides used in this work are listed in Additional file 6: Table S4. Following PCR, the PCR products were run on an agarose gel for confirmation of a single band amplification at the expected size. The threshold levels of each amplification were adjusted to the logarithmic part of the curve for determining a Ct value. Then the Ct values were normalized with those of β-actin to obtain the relative mRNA levels. The normalized data were analyzed using a Student’s t-test, and the confidence levels were displayed as p-values.

Total RNA from samples were reverse transcribed using Mir-X™ miRNA First-Strand Synthesis Kit (Clontech, Mountain View, CA, USA) and real-time PCR was performed using SYBR® Advantage® qPCR Premix (Clontech). PCR primers for miRNA-33a were prepared as follows: miRNA-33a forward, 5′-GTGCATTGTAGTTGCATTGCA-3′ [11 (link), 12 (link), 15 (link)], while the 3′ primer was included in the kit as a reverse primer. PCR was performed using a Rotor-Gene 6000 (Corbett Life Science, Concorde, NSW, Australia). As an internal control, a known amount of synthetic U6 snRNA was added to each sample, which was evaluated using Clontech Mir-S miRNA First Strand Synthesis and a SYBR RT-PCR kit. We calculated the ∆Ct of miR-33a in each sample using the internal control, U6 snRNA, by subtracting the Ct of U6 from the Ct of miR-33a. Then, we calculated the respective ∆∆Ct in each sample by subtracting the mean ∆Ct of the normal group from the ∆Ct of each sample. We calculated the 2^−∆∆Ct values for each sample and determined the mean value for each group. Finally, we compared the expression levels of miR-33a in plasma of three groups and in culture supernatant of five types of macrophages.

To ensure transcription levels of key genes were not influenced by PC4 status, cDNA was made from total mRNA using the RNeasy Mini Kit (Qiagen) and SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) according to manufacturer’s instructions. Quantitative PCR (qPCR) was performed on the LightCycler 1.5 (Roche) using the LightCycler FastStart DNA Master SYBR Green I (Roche) kit according to manufacturer’s instructions. Primers used were rbc9-rbc10 for the B-cell activating factor (BAFF) control, rbc22-rbc4 for Igλ, AI24-AI25 for AID, and rbc732-rbc585 and rbc265-rbc585 for GFP.
To evaluate PC4iresGFP transcript stability, cDNA was made as above from bulk or GFP sorted (positive and negative) populations, however the qPCR was performed on the ViiA 7 System (Applied Biosystems) using the SYBR Advantage qPCR Premix (Clontech) according to manufacturer’s instructions. Primers used were rbc9-rbc10 for the BAFF, AI24-AI25 for AID, rbc615-rbc616 for PC4 and rbc843-rbc844 for GFP.