Sybr fast universal 2x qpcr master mix

Plasma samples were thawed at room temperature, centrifuged to pellet any cell debris, and spectrophotometrically analyzed for oxyhemoglobin absorbance at 414 nm. The cut‐off level was set at 0.22 against water. Approximately 8.7% and 11.9% of healthy controls and PD plasma samples, respectively, were found hemolyzed and were discarded. miRNA extraction was performed using the NucleoSpin^® miRNA plasma kit from Macherey‐Nagel. About 1 μg of MS2 (Roche) RNA was added to improve miRNA yield during the extraction method. Polyadenylation and reverse transcription reactions were performed in triplicate for every sample. Similarly, qPCR was performed in triplicate on the Roche Lightcycler^® 96 using the SYBR FAST Universal 2X qPCR Master Mix from Kapa Biosystems. Stable reference (miR‐103a‐3p, miR‐191‐5p, miR‐425‐5p, miR‐223‐3p, miR‐423‐3p) and hemolysis (miR‐23a, miR‐451a) miRNA controls were included in the plasma analysis and were identical to previous study.¹⁴ Primer sequences can be found in Table S1. The relative expression level of miRNAs was calculated using the 2^−ΔΔCt method. Samples with a cycle threshold difference Δ(Ct_23α‐3p‐Ct_451α)> 5.5, a PCR‐based indicator of hemolysis, were excluded from the final analysis.

LNCaP cells were cross-linked for 10 minutes at room temperature with 0.75% formaldehyde-containing phosphate-buffered saline; cross-linking was stopped with phosphate-buffered saline–glycine (0.3 M final). Cells were resuspended in cell lysis buffer (50-mM Tris, pH 8.1, 1% SDS, 10-mM EDTA) and incubated for 10 minutes on ice. Lysates were then sonicated to obtain DNA fragments averaging 200 to 500 bp in length. Sonicated lysates were cleared by centrifugation and diluted in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2-mM EDTA, 20 mM Tris-HCl, pH 8.1, 167-mM NaCl) and immunoprecipitated overnight with 2 μg of indicated antibodies. Immunoprecipitated DNA was analyzed by qPCR (KAPA SYBR FAST Universal 2X qPCR Master Mix) together with 1% of the input chromatin. Specific primer pairs were designed to amplify the promoter region of the human androgen response element of the PSA gene: (5’-AACAGACCTACTCTGGAGGAACA -3’ and 5’-TCCAGGCTTGCTTACTGTCC-3’), and Nkx3.1 gene: (5’-ATCTGGGAGACTGGCAAAGA-3’ and 5’-GGCACTTCCTGAGCAAACTT-3’), which were designed to amplify one of the AR peak regions [53 (link)].

Total cellular RNA was extracted using Trizol (catalog no. 15596018) as per the manufacturer’s instructions. One microgram of total RNA was used for cDNA preparation using SuperScript IV reverse transcriptase kit (Catalog no. 18090200).
PCR was done using KAPA SYBR FAST Universal 2X qPCR Master Mix (Catalog no. KK4618). The following primer pairs were used for assaying overexpression of human Sirt4, forward (5′-CAGCGCTTCATCACCCTTTC-3′) and reverse (5′-CCTACGAAGTTTCTCGCCCA-3′), and normalized to mouse Actinb, forward (5′-GCATGGGTCAGAAGGATTCC-3′) and reverse (5′-ACGCAGCTCATTGTAGAAGG-3′).