Rotor gene q real time analyzer

Cells were lysed and total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. The RNA quality and concentration were estimated using denatured gel electrophoresis and spectrophotometry respectively. About 500 ng of the total RNA was reverse transcribed into cDNA using a reverse transcription kit (Fermentas, USA) with random hexamers for target genes. cDNA synthesis of miRNAs was undertaken using the Reverse Transcription System Kit (Promega, USA) with miR-specific stem-loop primers (Table 2). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using a 40 cycle PCR in Rotor-gene Q real-time analyzer (Corbett, Sydney, Australia). Each real-time PCR reaction contained 5 μl of 2×SYBR Premix Dimer EraserTM (TaKaRa, Japan), 3 pmol of forward and reverse primers respectively, 1 μl template of cDNA and dH₂O up to the final volume of 10 μl, followed by a melting curve analysis to confirm PCR specificity. The average threshold cycle was used for data analysis by Rotor-gene Q software (Corbett, Sydney, Australia). Gene expression levels were normalized against the expression of β-actin and Snord 47(U47) as internal controls for miRNA expression. The 2 ^−ΔΔCtmethod was employed to estimate the relative expression level of each gene. All reactions were run in triplicate.

LC3 and P62 genes were evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) at the mRNA level. NIH/3T3 cells were treated with normal, acidic, conditioned, and conditioned plus acidic media for 72 hours. Total RNA was extracted using the RNX-Plus kit (Sinaclon, Iran), and DNAse 1 treatment was applied. Random hexamer and oligo dt-primed cDNA synthesis was carried out using a cDNA synthesis kit (Takara, Japan). cDNA was used for 45-cycle RT-PCR in a Rotor-Gene Q real-time analyzer (Corbett, Australia) using the EvaGreen master mix. Each reaction was repeated three times, and the relative fold change in gene expression was quantified using the DDCt method. The β-actin gene was selected as an internal control. The duplicate experiments were repeated three times, and the mean ± SD was calculated. The primer sequences of the genes were provided as follows.
β-Actin
F: 5´-TGAAGATCAAGATCATTGCTCCTC- 3´
R: 5´-TCAGTAACAGTCCGCCTAGAAG- 3´
P62
F: 5´-TGTGGAACATGGAGGGAAGAG- 3´
R: 5´-TGTGCCTGTGCTGGAACTTTC- 3´
LC3
F: 5´-GACGGCTTCCTGTACATGGTTT- 3´
R: 5´-TGGAGTCTTACACAGCCATTGC- 3´