Paxgene blood rna kit ivd

RNA was extracted from frozen PAXgene blood RNA tube of psoriasis patients and control subjects. Total RNA was extracted using the PAXgene Blood RNA Kit IVD (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. To ensure complete DNA removal, on-column DNase digestion was performed and RNA was eluted in 15–20 μl RNase-free water.

RT-PCR sequencing was performed as previously described [28 (link)]. Briefly, we isolated RNA using the PAXgene Blood RNA Kit IVD (Qiagen, Hilden, Germany) and performed RT-PCR using the OneStep RT-PCR Kit (Qiagen). Primer sequences were as follows: Forward primer: 5′-CCTGGCGAATATGCTGTTCACATC-3′ and reverse primer: 5′-CGGGGTGTATGAGCATGCATATGT-3′. Amplicons were extracted from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and were subsequently sequenced. Sequence traces were assembled, aligned and analysed with the Seqman software (DNASTAR Lasergene, Madison, WI, USA).

Total RNA was extracted from the patient’s LCLs and peripheral blood using the High Pure RNA Isolation Kit (Roche, Basel, CH) and PAXgene Blood RNA Kit IVD (QIAGEN, Hilden, DE), respectively, according to the manufacturer’s instructions. cDNA was synthetized using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, CH), and RT-PCRs were prepared with KAPA2G Fast HotStart (KAPA Biosystems, Wilmington, MA). To verify the deletion from exons 11 to 14, RT-PCR primers 9F 5′-TTG​ATG​TAC​TGC​AGA​GAA​TGC-3′ and 16R 5′-GTG​TCT​TGG​CCA​ATG​AGA​TG-3’ were used. To assess the c.2778 + 83C>G effect, RT-PCR primers 27F 5′-AGC​TGC​TTA​TCT​CCA​GGC​C-3, 28R_+83 5′-CAA​ACC​CTA​GAC​TCA​GGA​CG-3,′ and 30R 5′-GGA​AAT​CCA​TCA​GTG​CGT​TG-3′3′ were designed within the predicted intronic retention and used in Savino et al. (1997) (link) to characterize the variant. RT-PCR amplicon extraction from 3% agarose gel was performed using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, DE) in line with the manufacturer’s instructions. To increase the performance and purify the extraction, the products obtained were further subjected to classical PCR with the same primer pair used for RT-PCR. All RT-PCR amplicons were purified and sequenced, as reported previously.

Total RNA was extracted from the patient’s LCLs and peripheral blood using the High Pure RNA Isolation Kit (Roche, Basel, CH) and PAXgene Blood RNA Kit IVD (QIAGEN, Hilden, DE), respectively, according to the manufacturer’s instructions. cDNA was synthetized using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, CH), and RT-PCRs were prepared with KAPA2G Fast HotStart (KAPA Biosystems, Wilmington, MA). To verify the deletion from exons 11 to 14, RT-PCR primers 9F 5′-TTG​ATG​TAC​TGC​AGA​GAA​TGC-3′ and 16R 5′-GTG​TCT​TGG​CCA​ATG​AGA​TG-3’ were used. To assess the c.2778 + 83C>G effect, RT-PCR primers 27F 5′-AGC​TGC​TTA​TCT​CCA​GGC​C-3, 28R_+83 5′-CAA​ACC​CTA​GAC​TCA​GGA​CG-3,′ and 30R 5′-GGA​AAT​CCA​TCA​GTG​CGT​TG-3′3′ were designed within the predicted intronic retention and used in Savino et al. (1997) (link) to characterize the variant. RT-PCR amplicon extraction from 3% agarose gel was performed using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, DE) in line with the manufacturer’s instructions. To increase the performance and purify the extraction, the products obtained were further subjected to classical PCR with the same primer pair used for RT-PCR. All RT-PCR amplicons were purified and sequenced, as reported previously.

RNA extraction was performed with a PAXgene Blood RNA kit IVD (QIAGEN, Hilden, Germany). The RNA-seq library was prepared with a TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA). The messenger RNA libraries were sequenced by paired-end 100 cycles on the HiSeq 2500 (Illumina). Low-quality bases in the raw reads were filtered out by FastQC ver 0.11.5 [16 ] and potentially existing sequencing adapters were trimmed with Skewer ver 0.2.2 [17 (link)]. The high-quality reads were mapped to the human reference genome hg19 (downloaded from UCSC genome browser, https://genome.ucsc.edu) by STAR ver 2.6 [18 (link)]. The gene expression level was quantified based on the aligned reads by Cufflinks package ver 2.2.1 [19 (link)]. The gene annotation of the reference genome was used as gene models and the expression values were calculated in Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) unit.