Hotfire polymerase

Protein A from Staphylococcus aureus (P3838), biotinylated native Protein A (P2165), and recombinant Protein A (P7837, expressed in E. coli), as well as human serum albumin (HSA, A9511), bovine serum albumin (BSA, A3059), human thrombin (89223 Fluka), and human immunoglobulin G (IgG, I4506) were purchased from Sigma-Aldrich (Germany). Human IgG-Fc fragment (009–0103) and human IgG-Fab fragment (009–0105) were from Rockland Immunochemicals, Inc. (Ireland). Recombinant Protein G and Protein L (Pierce 21193 and 21189, expressed in E. coli) were purchased from Fisher Scientific (Germany). Superparamagnetic Dynabeads M-270 Streptavidin (Strep-MB) were purchased from Invitrogen/Life Technologies (USA). According to the manufacturer’s instructions, these streptavidin-coated magnetic beads were used for immobilization of biotinylated native Protein A (P2165) as the target protein for aptamer selection to obtain Protein A-modified Strep-MB (Protein A/Strep-MB). PCR components like 10 × reaction buffer, 25 mM MgCl₂ and HOTFire polymerase were purchased from Solis BioDyne (Estonia). 100 mM stock solutions of dNTPs were from GE Healthcare (Germany).

In silico analysis of the TRKA gene structure and transcripts, reverse transcription and PCR methodology have been described before [69 (link)]. 5′ RACE experiments were conveyed with GeneRacer Kit (Invitrogen) according to the manufacturer’s protocol (Invitrogen). For PCR, HotFire polymerase from Solis Biodyne was used. All primers used in this study are listed in Additional file 2, TRKA ESTs identified with sequencing have been submitted and the corresponding GenBank accession numbers can be found in Additional file 3.

Total RNA from cells was purified with RNeasy Micro kit (Quiagen) as recommended by the manufacturer and treated with DNase I using DNA-free kit (Ambion). First-strand cDNA was synthesised from 5 μg of total RNA with Superscript III reverse transcriptase (Life Sciences) according to manufacturer’s recommendations. PCR reactions were performed with HotFire polymerase (Solis Biodyne) in a volume of 10 μl containing 1/80 of reverse transcription reaction as a template. Human BDNF 5′ exons’ specific primers have been described previously [12 (link)]. Rat BDNF 5′ exons’ specific primers have been described previously [11 (link)] and were used in combination with hRluc specific antisense primer 5′-GTA CTT GTA GTG ATC CAG GAG GCG AT-3′.