Qiamp rna blood mini kit

Skin tissue homogenate was snap frozen and RNA was isolated using RNA isolation kit (QI Amp RNA Blood Mini Kit, Qiagen, US). RNA was transcribed into cDNA using commercial kit (Qiagen, US, cat. no. 330401 RT2 First Strand Kit). The gene expression studies were performed using Qiagen signal transduction kit (Qiagen US, Cat No. PARN-014ZC). Gene expression of Ppard and Hmox1 was evaluated using TaqMan probes (Rn00565707_m1 and Rn00561387_m1 respectively) with Gapdh (Rn01775763_g1) as endogenous control. The reactions were carried out in a StepOne Plus real time PCR system (Applied Biosystems) using TaqMan universal master mix (Cat No. 4364338, Thermo Fisher) as per manufacturer's recommendations.

Total RNA isolation from bone marrow samples has been performed with the Qiamp RNA blood mini kit (Qiagen).

RNA was isolated from 1 × 10⁶ cells using the QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. One microgram of the total RNA was subjected to reverse transcription using the QuantiTec Reverse Transcription Kit (Qiagen, Hilden, Germany). Relative quantitative real-time PCR was performed to evaluate the expression levels of the HERV-H, -K, -P, and -W env genes in a 7500 real-time PCR system (Applied Biosystem, CA, USA) using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, with the primer sets shown in Supplementary Table S2. Forty cycles were performed for each real-time PCR assay, with the temperatures of annealing at 54 °C. All the samples were tested in triplicate and were compared to the mean expression of the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) and β-actin. Non-template controls were included for each primer pair. The quantification of the RNA expression was performed using the comparative Ct method, and the differences between the levels of env gene expression in the biological samples were calculated by relative quantification (RQ).

Total RNA was extracted from powdered livers of Pah-R261Q and WT mice (3-months-old; n = 5–6 per group) with the QIAmp RNA Blood Mini Kit (QIAGEN) and translated into cDNA using the Reverse Transcription System (Promega). Quantitative PCR was performed in standard triplicate assays for each mouse sample with 50 ng of cDNA using TaqMan technology, an ABI Prism 7700 sequence detector, and the TaqMan Universal PCR Master Mix from ABI. Detailed information about the specific transcript detection cannot be given, as we used ABI TaqMan Gene Expression Assays, which are under a proprietary license, and the exact primer and probe sequence are not disclosed. The ABI assay numbers for the indicated murine mRNA and NCBI nucleotide sequence numbers are summarized in Supplementary Table 4. Murine Gapdh-mRNA was included as a control for normalization and the analyses of the relative gene expression were performed based on the 2^−ΔΔCt method^{77 (link)}.

Peripheral blood mononuclear cells (PBMC) were collected by Ficoll-paque (GE Healthcare) purification and genomic DNA was extracted from PBMC of collected patients from KMUH. Total RNA was extracted by the method of QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany). RNA was reversely transcribed to cDNA with random primers and high capacity cDNA Archive kit (Applied Biosystems, Life Technologies, Waltham, MA, USA).