Faststart taq dna polymerase kit

RNA was extracted from 2.5 ml of blood using the PAXgene kit (Qiagen) and Reverse Transcriptase (RT) reactions were performed using the QuantiTect RT kit (Qiagen). Relative allelic expression of PIGY was measured by analysing cDNA using both Sanger sequencing and a 314 chip run on the Ion Torrent PGM platform. As the c.-540G>A variant is only 4 bp from the transcription start site, expression levels had to be measured indirectly using the T allele at rs3177413 (c.-222C>T) which familial transmission showed was in cis with c.-540G>A allele. RT-PCR primer sequences are shown in Table 1. The first round of amplification (25–30 cycles) and second round of amplification (15 cycles, with barcoding primers) were both performed with the FastStart Taq DNA polymerase kit (Roche). Samples were pooled, cleaned using Ampure beads, quantified using the BioAnalyser (Agilent) and diluted to 10 pM. Emulsion PCR and sequencing were carried out according to manufacturer instructions.
Quantitative PCR was performed using IQ SYBR Green Supermix (BIO-RAD) and the iQ5 Real-Time PCR Detection System (BIO-RAD) and the following primers: PIGY-V5-F 5′-AGGGATGTTCATCTCCAACCA-3′, PIGY-V5-R 5′-TGCGCATATCAGGCTTAGGA-3′, RPL30-F 5′-CAGACAAGGCAAAGCGAAAT-3′ and RPL30-R 5′-TGGACACCAGTTTTAGCCAAC-3′. PCRs were performed in triplicate, and the experiment was carried out three times.

In order to identify the chlamydial species, the samples that had tested positive to the real-time PCR were characterized by sequencing a 16S rRNA gene portion of 278 bp with the primers set described by Vicari et al. [43 ]. The FastStart™ Taq DNA Polymerase kit (Roche Diagnostics, Mannheim, Germany) was used with 5 µL of extracted DNA as template in a final volume of 25 µL with the following final concentrations: 2.5 mM of MgCl₂, 0.2 mM of each dNTPs and 0.4 µM of each primer. After the initial denaturation step at 95 °C for 15 min, a touchdown protocol was applied to 20 cycles at 95 °C for 30 s, a decreasing annealing temperature from 65 °C to 55 °C for 30 s and 72 °C for 30 s, followed by 25 cycles with the same conditions and annealing step at 55 °C. A final extension step at 72 °C for 5 min was performed. PCR-amplified segments were purified with ExoSAP-IT Express PCR Product Cleanup (Applied Biosystems, Foster City, CA, USA) and sequenced with Brilliant Dye™ Terminator Cycle Sequencing kit Big Dye^® Terminator v3.1 Cycle Sequencing (Nimagen, Nijmegen, The Netherlands) on a the ABI PRISM^® 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequencing data were subjected to BLAST search against the NCBI database to identify the most related sequences.

Participants were recruited at the IRCCS Mondino Foundation, Pavia (Italy). Plasma isolated from 6 AD, 9 PD, 6 sporadic ALS (SALS), 9 FTD patients were used (Table 4). All patients were screened for mutations using a customized panel of 176 genes associated to neurodegenerative and neuromuscular diseases by Next Generation Sequencing (Sure Select QXT Target Enrichment, Agilent Technology, Santa Clara, CA, USA). ALS and FTD patients were screened for C9orf72 using the FastStart Taq DNA Polymerase Kit (Roche, Basel, Switzerland).
Diagnosis of AD was based on criteria expressed by Aging-Alzheimer’s Association workgroups [61 (link)]. For PD and FTD patients Movement Disorder Society (MDS) clinical diagnostic criteria were used [62 (link),63 (link)]. ALS diagnosis was made according to the revised El Escorial Criteria [64 (link)].
A total of six age-matched healthy volunteers free from any pharmacological treatment were recruited at the Immunohematological and Transfusional Service IRCCS Foundation “San Matteo”, Pavia (Italy) and used as healthy controls (CTRs). All the subjects were assayed to rule out the presence of inflammatory diseases by white blood cell counts and subjects with WBCs >11 × 10⁹ were excluded from the study. Patients’ characteristics are reported in Table 4, Table 5, Table 6 and Table 7.